Methods and compositions for expanding cell populations

Abstract

The present invention relates to methods for expanding stem cell populations and other cell populations. More particularly, the present invention relates to methods and compositions for expanding stem cell and / or other cell populations, particularly hematopoietic stem cell populations.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is filed as a PCT application and claims priority to U.S. Patent Application No. 62 / 570,076, filed on October 9, 2017, and U.S. Patent Application No. 62 / 695,820, filed on July 9, 2018, which are incorporated herein by reference in their entireties. Field of the Invention

[0003] The present invention relates to methods and compositions for expanding cell populations, and particularly stem cell populations, such as hematopoietic stem cell populations.

[0004] background

[0005] Hematopoietic stem cells (HSCs) are clonogenic cells that have the properties of self-renewal (expansion) and multi-lineage potential to produce all types of mature blood cells. HSCs are responsible for hematopoiesis and undergo proliferation and differentiation to produce mature blood cells of various lineages while still maintaining their ability to self-renew. The ability to self-renew maintains the HSC population throughout the life cycle of an animal and enables HSCs to repopulate the bone marrow of lethally irradiated syngeneic hosts.

[0006] Early stage HSC development shows a hierarchical arrangement, starting from the long-term (LT-) HSC with extensive self-renewal capacity, followed by the amplification state corresponding to short-term (ST-) HSC (with limited self-renewal capacity) and proliferative multipotent progenitor (MPP) (with multipotential potential, but without self-renewal capacity).MPP is also the stage of sensitization or preparation for differentiation.MPP is differentiated and directed to become the common lymphoid progenitor (CLP) that produces all lymphoid lineages or the common myeloid progenitor (CMP) that produces all myeloid lineages.In this process, more primitive colony produces less primitive cell mass, and it can not produce more primitive cell mass.The intrinsic genetic program that controls these processes (comprising the multipotentiality, self-renewal and activation (or transient amplification) of HSC, and the pedigree orientation from MPP to CLP or CMP) is still unknown to a large extent.

[0007] In order to maintain a constant production of blood cells throughout an individual's life, HSCs located in the bone marrow niche (Zhang, J. et al. Nature 425, 836-841, 2003; Calvi, LM et al. Nature 425, 841-846, 2003; Kiel, MJ, et al. Cell 121, 1109-1121, 2005; Arai, F. et al. Cell 118, 149-161, 2004) must strike a balance between quiescence and activation so that the immediate needs of hematopoiesis are met while also ensuring long-term stem cell maintenance. In adults, the dynamic balance between the quiescent and activated states of stem cells is important for protecting HSCs from losing their self-renewal potential and supporting ongoing tissue regeneration (Li, L. and Xie, T. Annu. Rev. Cell. Dev. Biol. 21, 605-631, 2005). Excessive activation and expansion of stem cells are both risky in eventually exhausting the stem cell population and predisposing to tumorigenesis. Although some factors important for stem cell activation have been identified (Heissig, B. et al. Cell 109, 625-637, 2002), little is known about the molecular events that govern the transition between quiescence and activation.

[0008] HSCs are responsible for lifelong hematopoiesis under both steady-state and stress conditions, which depends on a precise balance between stem cell self-renewal and differentiation (Li et al. Science, 327:542-545, 2010; Weissman et al. Cell, 100:157-168, 2000). Therefore, HSC transplantation is a life-saving therapy for the treatment of a variety of diseases, including blood, immune and genetic diseases and cancer (Walasek et al. Annals of the New York Academy of Sciences 1266:138-150, 2012). However, HSC-based therapies may be primarily limited by the lack of HLA-matched donor bone marrow (BM). Allogeneic transplantation provides another approach, but graft-versus-host disease (GvHD) remains a lifelong challenge because of the many side effects of immunosuppressive drugs, such as delayed immune recovery and thrombotic microangiopathy (Sung et al. Stem Cells Translational Medicine, 2:25-32 (2013); Shlomchik et al. Nature Reviews. Immunology, 7:340-352, 2007). Transplantation of HSCs from hUCB reduces the risk of GvHD; however, the number of HSCs in hUCB is less than that in BM or active peripheral blood, which limits its application (Walasek et al. Annals of the New York Academy of Sciences, 1266:138-150, 2012). Targeting single molecules or pathways has been investigated for hUCB HSC expansion (Huang et al. Leukemia, 30:144-153, 2016; Boitano et al. Science, 329:1345-1348, 2010; Fares et al. Science, 345:1509-1512, 2014; Ansellem et al. Nature Medicine, 9:1423-1427, 2003; Antonchuk et al. Cell, 109:39-45, 2002; Rentas et al. Nature, 532:508-511, 2016; Himburg et al. Nature Medicine, 16:475-482, 2010; North et al. Nature, 447:1007-1011, 2007; Guo et al. Nature Medicine, 2018); Varnum-Finney et al. Nature Medicine, 6: 1278-1281, 2000; and Chou et al. Experimental Hematology, 41: 479-490e474, 2013).However, other approaches are being sought to target differentiation in favor of stem cell self-renewal (Zhao et al. Molecular Cell Biology, 18:31-42, 2017).

[0009] m 6 A is a ubiquitous internal modification in mRNA that regulates gene expression by regulating RNA processing, localization, translation, and ultimately decay, which is regulated by the "writing," "erasing," and "reading" of the marker (Roundtree et al. Cell, 169: 1187-1200, 2017; Li et al. Annual Review of Genomics and Human Genetics, 15: 127-150, 2014). Recent studies have elucidated the role of mRNA in gene expression. 6 A modification plays a role in the transition from endothelial cells to hematopoietic cells during stem cell fate determination and embryogenesis (Batista et al. Cell Stem Cell, 15:707-719, 2014; Geula et al. Science, 347:1002-1006, 2015; Yoon et al. Cell, 2017; Zhang et al. Nature, 549:273-276, 2017; Zhao et al. Nature, 542:475-478, 2017) and in the development of leukemia (Li et al. Cancer Cell, 31:127-141, 2017; Vu et al. Nature Medicine, 2017; Barbieri et al. Nature, 2017; Weng et al. Cell Stem Cell, 22:191-205e199, 2018). Interestingly, m 6 Defects in the A writing complexes Mettl3 and Mettl14 lead to different outcomes in different types of stem cells. For example, Mettl3 or Mettl14 KO promotes HSC differentiation (Vu et al. Nature Medicine, 2017; Weng et al. Cell Stem Cell, 22:191-205e199, 2018; Barbieri et al. Nature, 2017), while also leading to enhanced stem cell self-renewal and maintenance in mouse embryonic stem cells (mESCs) and embryonic neuronal stem cells (NSCs) (Batista et al. Cell Stem Cell, 15:707-719, 2014; Yoon et al. Cell, 2017). In addition, m 6 The physiological functions of A in stem cells and leukemia are mediated by different mechanisms. 6 A modifies mRNA encoding stem cell fate determinants6 A-mediated decay regulates stem cell fate determination (Batista et al. Cell Stem Cell, 15:707-719, 2014; Yoon et al. Cell, 2017), while in acute myeloid leukemia (AML), Mettl3 and Mettl14 promote leukemogenesis because m 6 A modification stabilizes the mRNA of oncogenes and / or increases their translation (Vu et al., Nature Medicine, 2017; Barbieri et al., Nature, 2017; Weng et al., Cell Stem Cell, 22:191-205e199, 2018). In addition, previous studies have reported that the leukemogenic functions of FTO and Mettl14 are independent of the YTHDF reader protein (Li et al., Cancer Cell, 31:127-141, 2017; Weng et al., Cell Stem Cell, 22:191-205e199, 2018).

[0010] Due to m 6 A RNA modification is regulated by the “writing,” “erasing,” and “reading” of markers (Wang et al., Nature, 505(7481):117-120, 2014), so the process of installing, recognizing, and removing such and other markers may have various effects on cells, development, and disease processes. For example, studies have shown that m 6 The A tag may serve as a key post-transcriptional modification to promote the initiation of microRNA (miRNA) biogenesis (Alarcon et al. Nature, 519(7544):482-485, 2015). Evidence also indicates that m 6 A RNA modification may be involved in the differentiation of stem cells into specific lineages (Batista, Cellstem Cell, 15(6):707-719, 2014; Zhang et al. Nature, 549(7671):273-276, 2017), as well as regulating gene expression (Dominissini et al. Nature 485(7397):201-206, 2012; Haussmann et al. Nature 540(7632):301-304, 2016). 6 The A transferase METTL3 has been identified as a regulator of terminating murine naive pluripotency (Geula et al., Science, 347(6225):1002-1006, 2015). 6A “script” protein METTL3 disrupts T cell homeostasis and differentiation (Li et al. Nature, 548(7667):338-342, 2017), and has been found to 6 A RNA methylation promotes XIST-mediated transcriptional repression (Patil et al. Nature, 537(7620):369-373, 2016). 6 A RNA modification has also been shown to regulate UV-induced DNA damage response (Xiang et al. Nature, 543(7646):573-576, 2017). Studies on the maternal to zygotic transition (MZT) in zebrafish have also shown that m 6 A role of mRNA methylation in transcriptome turnover and animal development (Zhao et al. Nature, 542(7642): 475-478, 2017). 6 A's biological function (Lence et al. Nature, 540(7632); 242-247, 2016), but in adult stem cells m 6 The function of A is largely unknown.

[0011] Hematopoietic stem cells (HSCs) in the bone marrow (BM) maintain homeostatic hematopoiesis throughout life and also support regeneration after bone marrow ablation (Weissman, 2000). Quiescent HSCs outperform proliferative HSCs in lethally irradiated mice, largely due to the quiescence that protects HSCs from DNA damage (Arai et al., 2004; Fleming et al., 1993; Wilson et al., 2008) (Walter et al., 2015). However, a recent study showed that DNA damage accumulation in HSCs is associated with widespread weakening of DNA repair and a response pathway that depends on HSC quiescence (Beerman et al., 2014). In fact, although most HSCs are in a quiescent state, they are sensitive to DNA damage caused by chemotherapeutic drugs such as 5-fluorouracil (5FU) (Lerner and Harrison, 1990). An unresolved question is how the hematopoietic system overcomes the consequences of bone marrow ablation. Given the remarkable heterogeneity of HSCs during development and in adults (Benveniste et al., 2010; Benz et al., 2012; Fleming et al., 1993; Morita et al., 2010; Zhou et al., 2016), the existence of a reserve HSC (rHSC) subpopulation has been proposed, characterized by drug resistance and the ability to regenerate a large number of HSCs to overcome stress-induced bone marrow ablation (Haug et al., 2008; Li and Clevers, 2010; Wilson et al., 2008). However, to date, functional evidence supporting the existence of rHSCs in the blood system has not been provided.

[0012] HSCs are maintained in a complex BM niche for their maintenance and regeneration (Li and Clevers, 2010; Mendelson and Frenette, 2014; Morrison and Scadden, 2014; Scadden, 2014; Schofield, 1978). Over the past few decades, multiple studies have uncovered the complexity of the HSC BM niche components, including: endosteal (inner bone surface) cells (Calvi et al., 2003; Zhang et al., 2003), sinusoidal endothelial cells (Hooper et al., 2009; Kiel et al., 2005), Cxcl12-enriched reticular (CAR) cells (Sugiyama et al., 2006), nestin-expressing mitogen-activated protein kinase (NAPK)-mediated cytotoxicity (NET) (Calvi et al., 2003; Zhang et al., 2003), and mitogen-activated protein kinase (MIT)-mediated cytotoxicity (MIT) (Kiel et al., 2005). + and NG2 + Perivascular cells (Kunisaki et al., 2013; Mendez-Ferrer et al., 2010), LepR + and Prx-1 +Mesenchymal stem and progenitor cells (Ding and Morrison, 2013; Ding et al., 2012; Greenbaum et al., 2013), non-myelinating Schwann cells (Yamazaki et al., 2011), and megakaryocytes (Bruns et al., 2014; Zhao et al., 2014). However, whether and how BM niche complexity contributes to the regulation of HSC heterogeneity remains largely unclear (Itkin et al., 2016). Furthermore, the initial HSC niche was initially identified as spindle-shaped N-cadherin in the endosteal membrane of the cancellous bone region. + (N-cad + ) osteoblast precursor cells (Calvi et al., 2003; Xie et al., 2009; Zhang et al., 2003), but N-cad + The nature and function of niche cells remain unclear.

[0013] Therefore, there is still a need to clarify and understand m 6 A and m 6 A mRNA pathway to provide an understanding of the molecular regulation of stem cell proliferation and differentiation. There remains a need for methods of expanding stem cell populations in vivo and ex vivo, and for methods of using such expanded stem cell populations therapeutically, such as by transplantation into suitable subjects.

[0014] Overview

[0015] In one embodiment of the present disclosure, a method for expanding a stem cell population is provided, wherein the stem cell population is obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow. The method comprises regulating N in the stem cell population. 6 -methyladenosine (m 6 A) mRNA modification pathways to expand stem cell numbers.

[0016] In another embodiment, a method for ex vivo expansion of a substantially undifferentiated stem cell population is provided, comprising regulating the N 6 -methyladenosine (m 6 A) mRNA modification pathway to expand the number of undifferentiated stem cells without significant differentiation of the stem cell population.

[0017] According to another embodiment, a method is provided for ex vivo expansion of a hematopoietic stem cell (HSC) population obtained from a tissue selected from the group consisting of peripheral blood, umbilical cord blood, and bone marrow, the method comprising regulating N in the HSC population. 6 -methyladenosine (m 6A) mRNA modification pathways to expand HSC populations to sufficient numbers for subsequent transplantation into subjects in need thereof while maintaining multi-lineage differentiation potential within the HSC population.

[0018] According to another embodiment, a method is provided for expanding hematopoietic stem cells (HSCs) ex vivo by at least 2-fold, said expanded HSCs being capable of reconstituting the HSC lineage when transplanted into a mammal in need thereof, the method comprising: 6 A deletion, replacement, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into stem cells, and the HSC population is cultured in an appropriate culture medium.

[0019] According to another embodiment, a kit is provided for expanding a population of hematopoietic stem cells (HSCs) for subsequent transplantation into a subject in need thereof, the kit comprising a method for converting a gene that results in expression of m 6 A system for introducing deletion, substitution, or expression-reducing mutations in genes encoding mRNA modification readers into an HSC population, and instructions for use thereof.

[0020] According to another embodiment, a kit is provided for expanding a hematopoietic stem cell population (HSC) for subsequent transplantation into a subject in need thereof, the kit comprising m 6 A. Inhibitors of mRNA modification readings, and instructions for their use.

[0021] In another embodiment, a method of administering hematopoietic stem cells (HSCs) to a subject in need thereof is provided, the method comprising: (a) administering a gene that results in expression of m 6 A. introducing a deletion, substitution, or expression-reducing mutation of a gene for a modified mRNA reader into a sample containing a population of HSCs; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the HSCs to the subject.

[0022] In another embodiment, a method of administering hematopoietic stem cells (HSCs) to a subject in need thereof is provided, the method comprising: (a) growing hematopoietic stem cells (HSCs) in a suitable culture medium in a medium containing 6 A. culturing a sample comprising a population of HSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 A. an inhibitor of mRNA modification readings; and (c) administering HSCs to the subject.

[0023] In another embodiment, a method of reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) causing expression of m6 A. introducing a deletion, substitution, or expression-reducing mutation of a gene for a modified mRNA reader into a sample containing a population of HSCs; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the HSCs to the subject.

[0024] In another embodiment, a method of reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) in a suitable culture medium, 6 A. culturing a sample comprising a population of HSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture. 6 A. an inhibitor of mRNA modification readings; and (c) administering HSCs to the subject.

[0025] In one embodiment, a method of expanding a hematopoietic cell (HSC) population is provided, comprising culturing the HSC population under conditions sufficient to result in at least a 2-fold expansion of the HSC population, wherein the expanded HSC population is suitable for transplantation into a mammal in need thereof.

[0026] In another embodiment, a method for expanding a hematopoietic stem cell (HSC) population is provided, comprising: (a) obtaining a tissue sample comprising an HSC population from a mammal; (b) expanding the HSC population from the sample in vitro, wherein: (i) the HSC population is expanded at least 2-fold; and (ii) the total colony-forming units of the expanded HSC population are increased at least 5-fold.

[0027] In another embodiment, a method for reconstituting a hematopoietic stem cell lineage in a subject in need thereof is provided, the method comprising: (a) obtaining a tissue sample comprising an HSC population from a mammal; (b) expanding the HSC population from the sample in vitro, wherein: (i) the HSC population is expanded at least 2-fold; (ii) the total colony-forming units of the expanded HSC population are increased at least 5-fold; and (c) transplanting the expanded HSC population into a subject in need thereof.

[0028] In another embodiment, a method is provided for expanding a hematopoietic stem cell population in a mammal in need of such expansion, the method comprising administering to the mammal a therapeutically effective amount of N 6 -methyladenosine (m 6 A) Modulators of mRNA modification pathways for a period of time sufficient to allow for at least a 2-fold expansion of the HSC population, resulting in the ability of HSCs to reconstitute hematopoietic lineages in mammals.

[0029] According to another embodiment, a method is provided for ex vivo expansion of a mesenchymal stem cell (MSC) population obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow, the method comprising regulating N in the MSC population. 6 -methyladenosine (m 6 A) mRNA modification pathways to expand MSC populations to sufficient numbers while maintaining multi-lineage differentiation potential in the MSC population sufficient for subsequent transplantation into a subject in need thereof.

[0030] According to another embodiment, there is provided a method for expanding mesenchymal stem cells (MSCs) at least 2-fold ex vivo, said expanded MSCs being capable of reconstituting the MSC lineage when transplanted into a mammal in need thereof, the method comprising: 6 A deletion, replacement, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into stem cells, and the MSC population is cultured in an appropriate culture medium.

[0031] According to another embodiment, there is provided a kit for expanding a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof, the kit comprising a method for causing expression of m 6 A system for introducing deletion, substitution, or expression-reducing mutations of genes encoding mRNA modification reads into a MSC population, and instructions for use thereof.

[0032] According to another embodiment, a kit for expanding a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof is provided, the kit comprising m 6 A. Inhibitors of mRNA modification readings, and instructions for their use.

[0033] In yet another embodiment, a method for administering mesenchymal stem cells (MSCs) to a subject in need thereof is provided, the method comprising: (a) administering a cell that results in expression of m 6 A method for the present invention relates to a method for the present invention to introduce a deletion, substitution, or expression-reducing mutation of a gene of a mRNA modification reader into a sample containing a population of MSCs; (b) culturing the sample in a suitable culture medium for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the MSCs to the subject.

[0034] In another embodiment, a method for administering mesenchymal stem cells (MSCs) to a subject in need thereof is provided, the method comprising: (a) in a suitable culture medium, 6 A. culturing a sample comprising a population of MSCs in the presence of an inhibitor of mRNA modification for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture;6 A. an inhibitor of mRNA modification readings; and (c) administering MSCs to the subject.

[0035] In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, the method comprising: (a) causing expression of m 6 A method for the present invention relates to a method for the present invention to introduce a deletion, substitution, or expression-reducing mutation of a gene of a mRNA modification reader into a sample containing a population of MSCs; (b) culturing the sample in a suitable culture medium for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the MSCs to the subject.

[0036] In another embodiment, a method of reconstituting bone marrow in a subject in need thereof is provided, the method comprising: (a) in a suitable culture medium, 6 A. culturing a sample comprising a population of MSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to allow the number of MSCs in the sample to expand to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 A. an inhibitor of mRNA modification readings; and (c) administering MSCs to the subject.

[0037] In one embodiment, a method for expanding a mesenchymal cell (MSC) population is provided, the method comprising culturing the MSC population under conditions sufficient to result in at least a 2-fold expansion of the MSC population, wherein the expanded MSC population is suitable for transplantation into a mammal in need thereof.

[0038] In another embodiment, a method for expanding a mesenchymal stem cell (MSC) population is provided, the method comprising: (a) obtaining a tissue sample comprising an MSC population from a mammal; (b) expanding the MSC population from the sample in vitro, wherein: (i) the MSC population is expanded at least 2-fold; and (ii) the total colony forming units of the expanded MSC population are increased at least 5-fold.

[0039] In another embodiment, a method of reconstituting a mesenchymal stem cell lineage in a subject in need thereof is provided, the method comprising: (a) obtaining a tissue sample comprising an MSC population from a mammal; (b) expanding the MSC population from the sample in vitro, wherein: (i) the MSC population is expanded at least 2-fold; (ii) the total colony forming units of the expanded MSC population are increased at least 5-fold; and (c) transplanting the expanded MSC population into a subject in need thereof.

[0040] In yet another embodiment, a method is provided for expanding a population of mesenchymal stem cells in a mammal in need of such expansion, the method comprising administering to the mammal a therapeutically effective amount of N 6 -methyladenosine (m6 A) Modulation of mRNA modifiers is sufficient to allow for at least a 2-fold expansion of the HSC population, resulting in HSCs having the ability to reconstitute the mesenchymal lineage in mammals.

[0041] According to another embodiment, a method is provided for ex vivo expansion of a mesenchymal stem cell (MSC) population obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow, the method comprising regulating N in the MSC population. 6 -methyladenosine (m 6 A) mRNA modification pathways to expand MSC populations to sufficient numbers while maintaining multi-lineage differentiation potential in the MSC population sufficient for subsequent transplantation into a subject in need thereof.

[0042] According to another embodiment, there is provided a method for expanding mesenchymal stem cells (MSCs) at least 2-fold ex vivo, the expanded MSCs being capable of reconstituting the MSC lineage when transplanted into a mammal in need thereof, the method comprising: 6 A deletion, replacement, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into stem cells, and the MSC population is cultured in an appropriate culture medium.

[0043] According to another embodiment, a kit for expanding a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof is provided, the kit comprising a method for causing expression of m 6 A system for introducing deletion, substitution, or expression-reducing mutations of genes encoding mRNA modification reads into a MSC population, and instructions for use thereof.

[0044] According to another embodiment, a kit for expanding a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof is provided, the kit comprising m 6 A. Inhibitors of mRNA modification readings, and instructions for their use.

[0045] In yet another embodiment, a method of administering mesenchymal stem cells (MSCs) to a subject in need thereof is provided, the method comprising: (a) causing the expression of m 6 A method for the present invention relates to a method for the present invention to introduce a deletion, substitution, or expression-reducing mutation of a gene of a mRNA modification reader into a sample containing a population of MSCs; (b) culturing the sample in a suitable culture medium for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the MSCs to the subject.

[0046] In another embodiment, a method for administering mesenchymal stem cells (MSCs) to a subject in need thereof is provided, the method comprising: (a) in a suitable culture medium,6 A. culturing a sample comprising a population of MSCs in the presence of an inhibitor of mRNA modification for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 A. an inhibitor of mRNA modification readings; and (c) administering MSCs to the subject.

[0047] In another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) causing expression of m 6 A method for the present invention relates to a method for the present invention to introduce a deletion, substitution, or expression-reducing mutation of a gene of a mRNA modification reader into a sample containing a population of MSCs; (b) culturing the sample in a suitable culture medium for a period of time sufficient to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the MSCs to the subject.

[0048] In another embodiment, a method of reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) in a suitable culture medium, 6 A. culturing a sample comprising a population of MSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to allow the number of MSCs in the sample to expand to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 A. an inhibitor of mRNA modification readings; and (c) administering MSCs to the subject.

[0049] In one embodiment, a method for expanding a mesenchymal cell (MSC) population is provided, comprising culturing the MSC population under conditions sufficient to result in at least a 2-fold expansion of the HSC population, wherein the expanded MSC population is suitable for transplantation into a mammal in need thereof.

[0050] In another embodiment, a method for expanding a population of mesenchymal stem cells (MSCs) is provided, comprising: (a) obtaining a tissue sample comprising a population of MSCs from a mammal; (b) expanding the MSC population from the sample in vitro, wherein: (i) the MSC population is expanded at least 2-fold.

[0051] In another embodiment, a method of reconstituting a mesenchymal stem cell lineage in a subject in need thereof is provided, the method comprising: (a) obtaining a tissue sample comprising a MSC population from a mammal; (b) expanding the MSC population from the sample in vitro, wherein: (i) the MSC population is expanded at least 2-fold; and (c) transplanting the expanded MSC population into a subject in need thereof.

[0052] In another embodiment, a method of expanding a population of mesenchymal stem cells in a mammal in need of such expansion is provided, comprising administering to the mammal a therapeutically effective amount of N6 -methyladenosine (m 6 A) Modulators of mRNA modification pathways are expressed for a period of time sufficient to allow for at least a 2-fold expansion of the MSC population, resulting in the ability of the MSCs to reconstitute the mesenchymal lineage in mammals.

[0053] In another embodiment, a method of isolating mesenchymal stem cells (MSCs) from a biological sample is provided, the method comprising contacting the biological sample having a population of MSCs with one or more N-Cadherin antibodies.

[0054] In another embodiment, there is provided an isolated population of mesenchymal stem cells as prepared by any of the methods described herein. According to yet another embodiment, there is provided an expanded isolated population of mesenchymal stem cells as prepared by any of the methods described herein.

[0055] According to another embodiment, a kit for isolating a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof is provided. The kit includes a system for contacting a biological sample containing MSCs with one or more N-Cadherin antibodies, and instructions for use thereof.

[0056] According to another embodiment, a method of administering mesenchymal stem cells (MSCs) to a subject in need thereof is provided. The method comprises: (a) isolating MSCs from a biological sample containing a population of mesenchymal stem cells (MSCs) by contacting the biological sample with one or more N-cadherin antibodies, and (b) administering the isolated MSCs to the subject.

[0057] According to another embodiment, a method of reconstituting bone marrow in a subject in need thereof is provided, comprising: (a) isolating mesenchymal stem cells (MSCs) from a biological sample comprising a population of MSCs by contacting the biological sample with one or more N-cadherin antibodies; and (b) administering the isolated MSCs to the subject.

[0058] According to yet another embodiment, a method for treating a subject in need of a transplant selected from a bone marrow transplant, a peripheral blood transplant, and an umbilical cord blood transplant is provided. The method comprises administering to the subject an isolated MSC population obtained by any of the methods described herein.

[0059] In another embodiment, a method for expanding a chimeric antigen receptor (CAR) T cell population prepared by modifying T cells obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow is provided. The method comprises regulating N in the CAR T cell population. 6 -methyladenosine (m 6 A) mRNA modification pathway to expand the number of CAR-T cells.

[0060] In another embodiment, a method for ex vivo expansion of a chimeric antigen receptor (CAR) T cell population is provided. The method comprises regulating the N 6 -methyladenosine (m 6 A) mRNA modification pathway to expand the number of CAR T cells.

[0061] According to another embodiment, a method for ex vivo expansion of a chimeric antigen receptor (CAR) T cell population prepared by modifying T cells obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow is provided. The method comprises regulating N in the CAR T cell population. 6 -methyladenosine (m 6 A) mRNA modification pathway to expand CAR T cell populations to sufficient quantities for subsequent transplantation into subjects in need thereof.

[0062] In another embodiment, a method for ex vivo expansion of chimeric antigen receptor (CAR) T cells is provided, wherein the expanded CAR T cells are capable of treating cancer and / or blood disorders when transplanted into a mammal in need thereof. The method comprises: 6 A deletion, substitution, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into a CART cell, and the CAR T cell population is cultured in an appropriate culture medium.

[0063] In another embodiment, a kit for expanding a population of chimeric antigen receptor (CAR) T cells (HSCs) for subsequent transplantation into a subject in need thereof is provided. The kit includes a method for causing expression of m 6 A system for introducing deletion, substitution, or expression-reducing mutations of a gene encoding a modified mRNA reader into a CAR T cell population, and instructions for use thereof.

[0064] In another embodiment, a method for administering a chimeric antigen receptor (CAR) T cell to a subject in need thereof is provided. The method comprises: (a) administering a chimeric antigen receptor (CAR) T cell to a subject in need thereof. 6 A. The invention relates to a method for introducing a deletion, substitution, or expression-reducing mutation of a gene of a mRNA modification reader into a sample containing a population of CAR T cells; (b) culturing the sample in a suitable culture medium for a period of time sufficient to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; and (c) administering the CAR T cells to the subject.

[0065] In another embodiment, a method of administering CAR T cells to a subject in need thereof is provided. The method comprises: (a) in a suitable culture medium, 6A. culturing a sample containing a population of CAR T cells in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 A. an inhibitor of mRNA modification readings; and (c) administering CAR T cells to the subject.

[0066] In another embodiment, a method for treating cancer and / or blood disorders in a subject in need thereof is provided. The method comprises: (a) causing expression of m 6 A. The method of claim 1 , wherein the deletion, substitution, or expression-reducing mutation of a gene of a modified mRNA reader is introduced into a sample containing a population of CAR T cells; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; and (c) administering the CAR T cells to the subject.

[0067] In another embodiment, a method for treating cancer and / or blood disorders in a subject in need thereof is provided. The method comprises: (a) in a suitable culture medium, 6 A. culturing a sample containing a population of CAR T cells in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 A. an inhibitor of mRNA modification readings; and (c) administering CAR T cells to the subject.

[0068] In one embodiment, a method for expanding a chimeric antigen receptor (CAR) T cell population is provided. The method includes culturing the CAR T cell population under conditions sufficient to cause the CAR T cell population to expand by at least 2-fold, wherein the expanded CAR T cell population is suitable for transplantation into a mammal in need thereof.

[0069] In another embodiment, a method for expanding a chimeric antigen receptor (CAR) T cell population is provided. The method includes: (a) obtaining a tissue sample comprising a T cell population from a mammal; (b) modifying the T cell population with a chimeric antigen receptor to provide a CAR T cell population; and (c) expanding the CAR T cell population from the sample in vitro, wherein: (i) the CAR T cell population expands at least 2-fold.

[0070] In another embodiment, a method for treating a subject with cancer and / or a blood disorder is provided. The method includes: (a) obtaining a tissue sample comprising a T cell population from a mammal; (b) modifying the T cell population with a chimeric antigen receptor (CAR) to form a CAR T cell population; (c) amplifying the CAR T cell population from the sample in vitro, wherein: (i) the CAR T cell population is expanded at least 2-fold; and (d) the expanded CAR T cell population is transplanted into the subject.

[0071] In another embodiment, a method of expanding a population of chimeric antigen receptor (CAR) T cells in a mammal in need of such expansion is provided. The method comprises administering to the mammal a therapeutically effective amount of N 6 -methyladenosine (m 6 A) A modulator of an mRNA modification pathway is administered to a mammal for a sufficient period of time to allow the CAR T cell population to expand at least 2-fold, such that the CAR T cells have the ability to treat cancer and / or blood disorders in a mammal.

[0072] In another embodiment, a method for treating a subject with a blood disorder is provided. The method comprises (a) obtaining a cell population selected from stem cells and T cells from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow; (b) optionally, where the cell population comprises T cells, modifying the T cells with a chimeric antigen receptor (CAR) to provide CAR T cells; (c) regulating N in the cells; 6 -methyladenosine (m 6 A) mRNA modification pathways to expand cell populations to expand cell number; and (d) transplanting the expanded cells into a subject to treat a blood disorder.

[0073] These and other aspects of the invention are further disclosed in the following detailed description and examples.

[0074] BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The following drawings constitute part of this specification and are included to further illustrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0076] Figures 1A-1H : Ythdf2 KO leads to elevated phenotypic HSCs in mice. Figure 1A Is to show Mx1-cre; Ythdf2 f / f Schematic representation of Ythdf2 deletion in HSPCs of conditional KO (cKO) mice. Figure 1B Shown are Western blots (left) and histograms (right) showing intracellular flow validation of Ythdf2 knockout in mouse HSPCs. Figure 1C Representative flow cytometric analysis of HSPCs in the BM from wt and Ythdf2 KO mice (n=5 per group). Figure 1D and 1E are bar graphs showing the frequency of total nucleated cells (TNC) of HSPCs (1D) and the absolute cell number of HSPCs (1E) in the BM from wt and Ythdf2KO mice (n=5 per group). Figure 1F is a bar graph showing the absolute number of BM TNCs from wt and Ythdf2 KO mice (n=5 per group). Figure 1G and 1H Bar graphs showing the absolute numbers of committed progenitor cells (1G) and lineage cells (1H) in the BM of wt and Ythdf2 KO mice (n=5 per group). Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0077] Figures 2A-2J : Ythdf2 KO leads to expansion of functional HSCs in mice. Figure 2A An experimental protocol for a limiting dilution transplantation assay (LDA) to determine the frequency of functional HSCs is described. Figure 2B It is a graph showing the primary LDA (n=10 per group) of CRU frequencies determined by ELDA (Limiting Dilution Analysis) 16 weeks after transplantation. Figure 2C Graph showing competitive reconstitution assays performed by transplanting 200K whole bone marrow (WBM) cells with 200K rescue cells into irradiated recipients (n=10 per group). Figure 2D and 2E are bar graphs showing the TNC frequency (2D) and absolute cell number (2E) of donor-derived HSPCs in the BM of transplant recipient mice as in (2C) (n=10 per group). Figure 2F and 2G is a bar graph showing the donor-derived (CD45.2 + ) Absolute cell numbers of committed progenitors (2F) and lineage cells (2G) (n=9-10 per group). Figure 2H Shown is a graph using secondary LDA to determine long-term CRU frequency by ELDA through 16 weeks after secondary transplantation (n=10). Figure 2I and 2JBar graphs showing quantification of functional mouse hematopoietic stem cells (HSCs) of total transplanted donor cells by transplantation assay and peripheral blood analysis (2I), and quantification of functional mouse HSCs by transplantation assay using the percentage of B, T, and myeloid lineage cells (2J) 4 weeks after transplantation, respectively. Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0078] Figures 3A-3I : Ythdf2 acts as m 6 A reader and regulates HSC gene expression by mediating mRNA decay. Figure 3A Figure 2 is a schematic diagram of the irCLIP-seq workflow. Figure 3B is a schematic diagram showing the Ythdf2 binding motifs identified by MEME. All irCLIP peaks were found in all three replicates. Figure 3C is a pie chart depicting the fraction of Ythdf2 binding peaks in each of the five transcript segments. Figure 3D is a graph showing GO enrichment analysis of Ythdf2 targets of intersecting genes from three Ythdf2 irCLIP-seq replicates. Figure 3E Shows the m 6 Representative trajectories of Tal1 for A-peak and Ythdf2irCLIP peaks, where m 6 A Coverage of immunoprecipitated and input fragments is indicated in red and grey, respectively, and Ythdf2irCLIP reads are highlighted in yellow. Figure 3F Figure 1 shows qPCR analysis of total mRNA from sorted LSK cells of wt and Ythdf2 KO mice. All Ct values ​​were first compared to the Actb control (non-m 6 The ratio (Ythdf2 KO vs. wt) was then calculated (n=3). Figure 3G Shown are representative images (left) and quantification of the staining intensity of wt (n=65) and Ythdf2KO (n=54) HSPCs by TAL1 (green) as histograms (right). Figure 3H Images are shown depicting fluorescent in situ hybridization of Tal1 mRNA (red) and fluorescent immunostaining of Dcp1a (P-body marker) (magenta), Ythdf2 (green) in wt and Ythdf2 KO HSPCs, with arrows indicating colocalized staining. Scale bar, 5 μm. Figure 3IQuantification of colocalization of Tal1 mRNA and DCP1a in sorted LSK cells from wt and Ythdf2KO mice is shown. Percentages represent the average frequency of Tal1 mRNA colocalized with DCP1a per LSK cell relative to the total Tal1 mRNA level (n = 9-17). Data are shown as mean ± sem. Unpaired t-test.

[0079] Figures 4A-4I :Through m 6 A-seq and RNA-seq analyses determined the role of YTHDF2 in human umbilical cord blood HSCs. Figure 4A Shown are the collective gene profiles depicting sequence coverage in windows around TSS (left) and stop codons (right), where m 6 Coverage of AIP and control (input) fragments are indicated in red and grey, respectively. Figure 4B Depicted are m in each of the five non-overlapping transcript segments. 6 A. Pie chart of peak scores. Figure 4C Depicted is a Venn diagram showing the common and unique m 6 A marked gene. Figure 4D Describes mRNAs common to mouse and hUCB HSPCs 6 A. GO enrichment analysis of the labeled transcripts. Figure 4E Shows the m 6 A Representative locus of HOXB4, where the color code is Figure 4A The same as in . Figure 4F hUCB CD34 + Schematic diagram of RNA-seq for lentivirus-mediated YTHDF2 KD in HSPCs. Figure 4G Control and YTHDF2 KD hUCB CD34 + Cellular m 6 A marker gene (purple line) and non-m 6 A. Cumulative distribution of log2 (fold change) of marker genes (black line). Figure 4H Representative coverage plots from RNA-seq analysis are shown, showing comparison with control hUCB CD34 + Compared with cells, YTHDF2 KD cells 6 A marker gene HOXB4 but not m 6 A marker gene ACTB has increased read counts. And, Figure 4I is a bar graph showing the difference between control and YTHDF2 KD hUCB CD34 + Non-m related to stem cell self-renewal 6A-labeled ACTB (as a control) and m 6 A. Relative mRNA expression levels of the labeled transcription factors. RPKM from RNA-seq analysis was normalized to the control. Adjusted P values ​​are shown. ns, not significant.

[0080] Figures 5A-5F :YTHDF2 KD promotes the in vitro expansion of human umbilical cord blood HSCs. Figures 5A-5B Bar graphs depicting the fold changes in frequency (5A) and absolute number (5B) of the indicated cells in YTHDF2 KD relative to control cells after 7 days of culture. Figure 5C It is shown that the transduced CD34 + CD38 - Histogram of CFU output by hUCB cells and images of CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (GEMM) (scale bar, 200 μm). Figure 5D Images containing burst-forming units-erythroid (BFU-E) (left) and colony-forming units-granulocytes / macrophages (CFU-GM) (right) from 7-day culture of control or YTHDF2 KD hUCB cells (scale bar (200 μm), where each graph uses two replicates of an independent umbilical cord blood sample). Figure 5E is a bar graph showing the expression of annexin V in transduced CD34 + CD34 in 7-day culture of hUCB cells + CD38 - Apoptosis analysis of cells (n=3 independent CB samples). Figure 5F Figure 2 is a bar graph showing the fold change of Tthdf2 knockdown (KD) relative to control in the indicated human umbilical cord blood HSCs (n=3 individual human samples), where lentivirus was used to deliver control shRNA or human Ythdf2 shRNA to sorted CD34 + CD38 - In umbilical cord blood HSCs. Dashed lines indicate 95% confidence intervals. Data are shown as mean ± SEM. Unpaired t-test. ns, not significant.

[0081] Figures 6A-6G :YTHDF2 KD promotes the expansion of functional long-term HSCs from human umbilical cord blood. Figure 6A is a diagram showing an experimental protocol for determining the frequency of HSCs following in vivo expansion. Figure 6B Includes hCD45 from naive recipient mice that received the highest two cell doses + GFP + Representative flow chart of reconstruction. hCD45 = human CD45. Figure 6CFigure 2 shows the expression of hCD45 in BM from naive recipient mice (n=8) that received the highest two doses. + GFP + Implanted diagram; Figure 6D Graph showing HSC frequencies determined by initial LDA. Dashed lines indicate 95% confidence intervals. Figure 6E Includes hCD45 from secondary recipient mice that received the highest two cell doses + GFP + Representative flow chart of reconstruction. Figure 6F Figure 2 shows the expression of hCD45 in BM from secondary recipient mice (n=6) that received the highest two doses. + GFP + Implanted diagram. Figure 6G Graph showing HSC frequencies determined by quadratic LDA. Dashed lines indicate 95% confidence intervals. Data are shown as mean ± SEM. Unpaired t-test. ns, not significant.

[0082] Figures 7A-7I : Ythdf2 KO HSCs showed no signs of lineage bias or differences in quiescence and homing capacity but displayed a lower apoptosis rate. Figure 7A is a bar graph showing the Mx1-cre without pI:pC injection - ;Ythdf2 f / f and Mx1-cre + ;Ythdf2 f / f The absolute cell number of HSPCs in the BM of mice (n=3 per group). Figure 7B is a bar graph showing cell cycle analysis of HSPCs in wt (n=3) and Ythdf2 KO (n=4) mice. Figure 7C is a bar graph showing apoptosis analysis of BM HSPCs in wt and Ythdf2 KO mice (n=5 per group). Figure 7D Images and weights of spleens from wt and Ythdf2 KO mice are shown. Figures 7E-7H Figure 2 shows the expression of TNC(7E), LSK, and CD48 in the spleen of wt (n=3) and Ythdf2 KO (n=4) mice. - CD150 + Bar graphs of absolute numbers of HSCs (7F), committed progenitors (7G), and lineage cells (7H). Figure 7I is a bar graph showing the homing ability of wt and Ythdf2KO cells, which was obtained by adding 1×10 6This was determined by transplanting CFDA-SE-labeled BM cells into lethally irradiated mice. 18 hours later, BM homing events were analyzed (n = 6 mice per group). Data are shown as mean ± SEM. Unpaired t-test. ns, not significant.

[0083] Figures 8A-8F : Recipient mice transplanted with Ythdf2 KO BM showed no lineage changes or defects 16 weeks after transplantation. Figures 8A to 8C is a bar graph depicting the donor-derived (CD45.2 + ) Absolute cell numbers of TNC (8A), committed progenitor cells (8B), and lineage cells (8C) (n=7-10 per group). Figures 8D to 8F is a bar graph depicting the donor-derived (CD45.2 + ) Absolute cell numbers of TNC (8D), committed progenitor cells (8E), and lineage cells (8F) (n = 7-10 per group). Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0084] Figures 9A-9K : Ythdf2 KO has long-term effects on the in vivo expansion of mouse HSCs without causing lineage bias. Figure 9A is a schematic diagram showing analysis of BM and spleen collected from wt and Ythdf2 KO mice by flow cytometry 5-7 months after pI:pC induction. Figures 9B to 9E is a bar graph showing the TNC ( Figure 9B )、HSPC( Figure 9C ), committed progenitor cells ( Figure 9D ) and lineage cells ( Figure 9E ) of the mice (n=4-7 mice per group). Figure 9F is a bar graph showing the weights of spleens from wt and Ythdf2 KO mice 5-7 months after induction (n=4-7 mice per group). Figures 9G to 9J is a bar graph showing the expression of TNC ( Figure 9G ), LSK CD48 - CD150 + HSC( Figure 9H ), committed progenitor cells ( Figure 9I ) and lineage cells ( Figure 9J ) of the mice (n=4-7 mice per group). Figure 9KFigure 5 shows 75k WBM from WT and Ythdf2 KO mice transplanted with 200k rescue cells into lethally irradiated recipients 5 months after pI:pC injection. Peripheral blood from transplant recipients was analyzed every 4 weeks after transplantation to determine the donor-derived graft (n = 10 per group). Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0085] Figures 10A-10D :m in mouse HSPC 6 Molecular characterization of A modifications. Figure 10A The collective gene profiles depicting sequence coverage in windows around TSS (top) and stop codons (bottom) are shown, where m 6 Coverage of AIP and control (input) fragments are indicated in red and grey, respectively. Figure 10B Pie charts are shown showing the m in each of the five transcript segments. 6 The score of peak A. Figure 10C Shows the presence of m in each segment in mouse HPSCs 6 A. Graph showing the fraction of genes in the peak as a function of expression level. Figure 10D Shown are m 6 A-labeled mRNA and non-m 6 A. Graph of the degradation rate of labeled mRNA.

[0086] Figures 11A-11E : Defining Ythdf2 function in mouse HSPCs by irCLIP-seq. Figure 11A is a graph showing immunoprecipitation of Ythdf2 in control or Flag-Ythdf2-overexpressing HPC7 cells. Figure 11B irCLIP membrane images showing IR800-labeled RNA-Ythdf2 complexes, where the red box indicates the RNA-Ythdf2 complexes collected for library construction, and a sample without UV cross-linking was used as a control. Figure 11C is a Venn diagram showing the intersecting genes identified in three independent Ythdf2ir CLIP-seq experiments. Figure 11D It is shown that Ythdf2 binds to the target and m 6 A. Venn diagram of the overlap of labeled mRNAs. Figure 11E Shows the m 6 Representative trajectories of Gata2 with A peak and Ythdf2irCLIP peak, where m 6 A Coverage of immunoprecipitated and input fragments is indicated in red and grey, respectively, and Ythdf2irCLIP reads are highlighted in yellow.

[0087] Figures 12A-12F :Ythdf2 KO increases m 6 A-marker mRNA expression contributes to HSC expansion. Figure 12A The bar graph shows the BM LSK Flk2 from 15,000 sorted - Total RNA was extracted from cells. Figure 12B wt and Ythdf2 KOLin are shown - m in cells (n=6) 6 A Quantification of RNA methylation. Figure 12C Shown are quantification (right) and histograms (left) showing intracellular flow cytometric validation of increased expression of TAL1, GATA2, RUNX1, and STAT5 in Ythdf2 KO LT-HSCs compared with wt LT-HSCs (n = 3 mice per group); Figure 12D Shown are fluorescence in situ hybridization for Gata2 mRNA (red) and fluorescent immunostaining for Dcp1a (P-body marker) (magenta) and Ythdf2 (green) in wt and Ythdf2 KO HSPCs. Arrows indicate colocalized staining. Scale bar, 5 μm. Figure 12E Quantification of colocalization of Gata2 mRNA and DCP1a in sorted LSK cells from wt and Ythdf2 KO mice is shown. Percentages represent the average frequency of Gata2 mRNA colocalized with DCP1a relative to total Gata2 mRNA levels per LSK cell (n=12-20). Figure 12F It showed that 4 weeks after transplantation, CD45 + GFP in the group + Percentage of cells (n=10). Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0088] Figures 13A-13G :YTHDF2 regulates the expression of transcription factors involved in stem cell self-renewal in human umbilical cord blood stem cells. Figure 13A is a Venn diagram showing the results of three independent m 6 Intersection genes identified in A-seq experiments. Figure 13B A pie chart is shown showing the 6 A. Percentage of mRNA and non-coding RNA in peak. Figure 13C is a bar graph showing the expression of CD34 in hUCB + Cells have m 6 A. GO enrichment analysis of modified transcription factors. Figure 13D Shows GFP in sorting +Images of Western blots of YTHDF2 (top) and β-actin (bottom) in control and YTHDF2 KD hUCB cells, showing the knockdown efficiency of YTHDF2. Figure 13E Control and YTHDF2 KD hUCB CD34 are shown + Bar graph (left) and representative trajectory graph (right) of YTHDF2 expression levels from RNA-seq analysis of cells, showing the knockdown efficiency of YTHDF2. Figures 13F to 13G Is with m 6 Representative trajectory plots (top) and representative coverage plots (bottom) of the indicated transcription factors for peak A. Adjusted P values ​​are indicated.

[0089] Figures 14A-14C : YTHDF2 KD in hUCB cells leads to HSC expansion without altering lineage output. Figure 14A Depicts the expression of GFP in control and YTHDF2 KD hUCB cells after 7 days of culture. + CD34 + CD38 - CD45RA - EPCR + Representative flow cytometry plots of HSCs. Figure 14B Depicted are confirmation of YTHDF2 protein knockdown and overexpression in transduced HeLa cells. Figure 14C CD34 transduced with YTHDF2 OE and control from day 10 cultures are depicted. + CD38 - CFU generated by CB (n = 3 independent human samples). Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0090] Figures 15A-15D : YTHDF2 KD in hUCB cells leads to HSC expansion without altering lineage output. Figure 15A Including hCD45 in BM of primary NSG recipients + GFP + Representative flow cytometry plots of monocytes, megakaryocytes (MK cells), B cells, and erythrocytes. Figure 15B and 15C is a bar graph depicting hCD45 from naive NSG recipients at 10 weeks post-transplantation. + GFP + ( Figure 15B ) and total CD45 + ( Figure 15C ) Percentage of lineage cells in BM cells (n=13-15). Figure 15Dis a bar graph depicting total CD45 from secondary NSG recipients at 12 weeks post-transplantation. + Summary of lineage chimerism in human donor-derived BM cells (n=6). Data are shown as mean ± sem. Unpaired t-test. ns, not significant.

[0091] Figure 16 : In vivo expansion of mesenchymal stem cells. Figure 16 is a bar graph showing the frequency of N-Cad+CD105+ in wt Ythdf2 and Ythdf2 knockout (KO) TNC.

[0092] Figures 17A-17J : Functional characterization of rHSC populations. Figure 17A Schematic diagram of FACS sorting of rHSCs, pHSCs, ST-HSCs and MPPs. Figure 17B Quantification of rHSC and pHSC function by transplantation assay is shown.PB analysis of total transplanted donor cells at the indicated weeks post-transplantation, and percentages of donor-derived B, T, and myeloid lineage cells at 20 weeks post-transplantation (n=10 mice per group). Figure 17C Donor-derived cells from rHSC- or pHSC-transplanted mice 40 weeks after transplantation are shown. Figure 17D : H2B-GFP labeled retained cells in rHSCs and pHSCs 130 days after chasing (n=4 mice per group). Figure 17E Cell cycle gene expression in rHSCs and pHSCs is shown (n=3 replicates from 20 mice). Figure 17F Recipients of rHSC and pHSC transplants were shown to receive 5FU injections 4 weeks after transplantation as indicated. PB analysis of donor transplanted cells was performed at the indicated weeks after transplantation. The percentages of donor-derived B, T, and myeloid lineage cells at 20 weeks after transplantation are shown (n=10 mice per group). Figure 17G Shown are rHSCs and pHSCs (pool from 15 mice) at day 3 after 5FU treatment. Figure 17H DNA damage gene expression in rHSCs and pHSCs is shown (n=3 replicates from 20 mice). Figure 17I DNA damage genes in rHSCs from control mice and mice on day 3 after 5FU are shown (n=20 in the control mouse group, n=40 in the D3 5FU mouse group). *P<0.05, **P<0.01, ***P<0.001. Error bars, sem. Figure 17J Heatmaps of stress response genes in rHSCs, pHSCs, and 5FU rHSCs are shown.

[0093] Figures 18A-18I: rHSCs are located in the endosteal region of the BM niche. Figure 18A Representative whole-mount images of mouse sternal bone marrow (BM) are shown, including bone (white, generated by second harmonic generation, SHG), MK (yellow, distinguished by size, morphology and CD41 expression). Green arrows indicate phenotypic Lin – CD48 – CD41 – CD150 + CD49b – rHSC, white arrows indicate phenotype Lin – CD48 – CD41 – CD150 + CD49b + pHSC. Figure 18B Representative images of rHSC, pHSC, and 5FU rHSC are shown. White arrows indicate phenotype Lin – CD48 – CD41 – CD150 + CD49b – rHSC, phenotypic Lin – CD48 – CD41 – CD150 + CD49b + pHSC and phenotypic Lin – CD48 – CD41 – CD150 + CD49b – 5FU rHSCs. Figure 18C -E shows the relative distances between rHSCs, pHSCs, and 5FU rHSCs and blood vessels, MKs, or bones (n=144 rHSCs, n=685 pHSCs, n=707 5FU rHSCs). Figure 18F Figure 3 shows healthy, apoptotic, and dead VE-Cad cells in control mice and mice treated with 5FU one day after treatment. + CD31 + The absolute number of vascular cells. Figure 18F -G shows Annexin V in central bone marrow (CM) from control mice and mice 1 day after 5FU treatment + SytoxG ─ Apoptotic Ve-Cad + CD31 + N-cad-tomato in endothelial cells or CM and bone +Absolute cell numbers. N = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, sem. Figure 18H -I shows N-cad-tomato expression in control mice and mice 3 days after 5FU treatment + and Ve-Cad + CD31 + Representative images of blood vessels.

[0094] Figures 19A-19J :N-cad + Cells maintain functional HSCs in the BM niche. Figure 19A showed that N-cad-CreER T DT regimen for iDTR mice used in the experiments shown in BG. D indicates date (e.g., D0 indicates day 0). Figure 19B N-cad-CreER T ; Tomato in iDTR mice + N-cad + Cell ablation efficiency. Figure 19C -D shows flow cytometry analysis to determine the N-cad-CreER from TMX and DT injection T ; Absolute numbers of total nucleated cells (TNC) and HSPCs in the bone marrow (BM) of iDTR mice (n=5 mice per group). Figure 19E -H shows quantification of functional HSCs by transplantation assay in primary 1° and secondary 2° transplants. Total BM cells from PB analysis of total transplanted donor cells at the indicated weeks post-transplantation, and the percentages of donor-derived B, T, and myeloid lineage cells at 16 weeks post-transplantation (n=10 mice per group). Figure 19I -J shows flow cytometric analysis to determine the N-cad-CreER from TMX and DT injection T ;SCFf / f(N-cad-CreER T _;SCFf / f, n=4 mice, N-cad-CreER T+ ; SCFf / f, n=6 mice) and N-cad-CreER T+ ; Cxcl12f / f mice (N-cad-CreER T _; Cxcl12f / f, n = 3 mice, N-cad-CreER T+ The absolute number of HSPCs in the bone marrow (BM) of Cxcl12f / f mice (*P<0.05, **P<0.01, ***P<0.001. Error bars, sem).

[0095] Figures 20A-20K : Transcriptome analysis of hematopoietic and niche cells. Figure 20A -B shows the Pearson distance tree and PCA analysis of hematopoietic stem and progenitor cells (HSPCs). Figure 20C -D shows the Pearson distance tree and PCA analysis of BM niche cells. Figure 20E The expression of HSC signature genes in hematopoietic stem and progenitor cells is shown. Figure 20F The expression of BM niche signature genes in niche cells is shown. Figure 20G Term analysis of BM niche cells is shown. Figure 20H The expression of stromal cell developmental genes in niche cells from the intraperiosteal and perivascular regions is shown. Figure 20I Shown is the target of N-cad-CreER after 3 TMX injections T ;R26-tdT;Nestin-GFP mice and N-cad-CreER after 3 TMX injections T Lineage tracing of ;R26-ZsG;Cxcl12-DsR mice. Figure 20J Shown are enzymatically digested bone marrow cells from N-cad-CreERT;R26-tdT mice following TMX injection. LepR and Pdfgrα were stained with antibodies, shown as red peaks. Isotype controls are shown as gray peaks. Figure 20K CFU-F activity in niche cells from the intraperiosteal and perivascular areas is shown.

[0096] Figures 21A-21G :N-cad + In vitro differentiation potential and localization of source cells. Figure 21A It is an experimental design. Figure 21B Shown is a viable CFU-F colony cultured from enzymatically digested bone marrow, with one colony under high magnification. Cell Trace TM and Tomato + Stained cells. Figure 21C -E shows the origin of N-cad-CreER T In vitro differentiation of stromal cells from R26-tdT mice: Live tdTomato cells 21 days after osteogenic differentiation + Alkaline phosphatase staining of cells and cultures (21C); Live Tomato 21 days after adipogenic differentiation + Cell and Oil Red O lipid staining (21D); Tomato + After 21 days of chondrogenic differentiation of cells, chondrocytes were stained with aggrecan antibody and toluidine blue (21E). Figure 21F -G shows (FG) experimental design. Figure 21HShown are the localizations of cells derived from N-cad-CreERT;R26-tdT mice 6 hours, 14 hours, 24 hours, 1 week, and 4 weeks after TMX injection. Figure 21I Figure 3 shows the Tomato counts in trabecular bone, cortical bone, and central bone marrow at 1, 2, 4, and 6 weeks after TMX injection. + Percentage of cells (n=2 mice in the 1-week and 2-week groups, n=3 mice in the 4-week and 6-week groups. *P<0.05, **P<0.01, ***P<0.001. Error bars, sem).

[0097] Figures 22A-22K :N-cad in adult mice + Stromal cells give rise to osteoblasts and adipocytes. Figure 22A N-cad-CreER from different time points after TMX injection is shown T Representative femoral sections of R26-tdT;Col2.3-GFP mice showing the anatomical distribution and increased Tomato + Col2.3-GFP + Osteoblast generation. Scale bar, 100 μm. Figure 22B -C shows N-cad-CreER in regions i, ii, iii, iv, v, and vi at early time points after TMX at 6 h (22B) and 14 h (22C). T High-power images of R26-tdT;Col2.3-GFP mice. The open arrows show the tomato folds at 6 and 14 hours after TMX. + Col2.3GFP + Osteoblasts (yellow cells), while solid arrows indicate recombined N-cad (green) in trabecular (ii, iv, v) and cortical (iii, vi) regions. These cells are potentially undifferentiated as indicated by the absence of Col2.3-GFP. Scale bar, 50 μm. Figure 22D -E shows image quantification, which shows that Tomato in trabecular (22D) and compact bone (22E) at 6 hours, 14 hours, 24 hours, 2 weeks and 4 weeks after TMX injection + Col2.3-GFP + and Tomato + Col2.3-GFP ─ The percentage of cells. Figure 22F Image quantification is shown, which compares the potential undifferentiated Tomato between trabecular bone and compact bone + Col2.3-GFP ─*P<0.05, **P<0.01, ***P<0.001. Error bars, sem. Figure 22G N-cad-CreER 4 weeks after TMX injection is shown T Representative images of trabecular bone (TB) and cortical bone (CB) in R26-tdT;Col2.3-GFP mice. Scale bar, 20 μm. Figure 22H N-cad-CreER stained with perilipin 4 weeks after TMX injection is shown. T Representative low-power and high-power images of femur sections from R26-tdT mice. Solid arrows indicate N-cadherin-derived bone marrow in the periosteum (i), bone marrow near the trabecular bone region (ii), and central bone marrow (iii). + MSC Tomato + perilipin + . Figure 22I BODIPY staining is shown, which shows that Tomato + perilipin + Lipid droplets (green) within adipocytes (arrows). Scale bar, 20 μm. Figure 22J -K shows Tomato in trabecular bone (22J) and periosteum (22K) at 6 hours, 14 hours, 24 hours, 2 weeks, and 4 weeks after TMX injection + perilipin + Image quantification of adipocytes. (*P<0.05, **P<0.01, ***P<0.001. Error bars, SEM).

[0098] Figures 23A-23J :N-cad + MSCs give rise to chondrocytes during development and after injury. Figure 23A It is an experimental design. Figure 23B Figure 3 shows that tomato colocalized with aggrecan+chondrocytes in the rib at E14.5 (2 days after TMX induction). + . Figure 23C A diagram illustrating chondrocyte development in the femur is shown. Figure 23D Two-day-old N-cad-CreER induced by TMX at E12.5 is shown T ; Representative femoral sections of R26-tdT mice. Note that Tomato + Cells produce aggrecan at the joint surface (i) and in developing secondary ossification centers (ii) + Chondrocytes. Arrows point to Tomato + Aggrecan + cell. Figure 23EA 10-month-old N-cad-CreER was shown to be induced in TMX at E12.5. T ; Representative femoral sections from R26-tdT mice. Tomato from early embryonic + perilipin + Fat cells. Figure 23F Two-month-old N-cad-CreER induced in TMX at E12.5 is shown T ; Representative femoral sections of R26-tdT mice. Tomato in early embryonic stage + Cells differentiated into osteopontin+ hypertrophic chondrocytes (i and ii) and osteoblasts (iii, iv, v, and vi); Figure 23G N-cad-CreER is shown for TMX induction at E12.5 T ; Experimental design of femoral groove injury in R26-tdT mice. Figure 23H The expression of tomato in control mice and mice 3 weeks after knee cartilage injury is shown. + Quantification of chondrocytes. Figure 23I N-cad-CreER without cartilage damage or 3 weeks after cartilage damage T ; Representative sections of the distal femur of R26-tdT mice. Note that the tomato aggregates on the knee surface of the control mouse (i). + Aggrecan + The cells increased significantly in the corresponding area of ​​mice (ii) 3 weeks after knee cartilage injury. Figure 23J AHO staining of the sections in Figure 6H is shown, showing Alcian blue-positive chondrocytes in the control (i) and in the site of cartilage damage (ii).

[0099] Figures 24A-24K : BrdU assay after 5FU and N-cad reporter mouse line. Figure 24A -F shows representative images showing BrdU in mouse femurs at days 0, 2, 3, 4, and 6 after 5FU treatment + cells. Note that BrdU + The cells decreased on day 2 after 5FU and reactivated near bone (red dashed line) or vascular / adipose structures (green dashed line) on days 3 to 6 after 5FU. Figure 24G BrdU + Dynamic model of the cell. Figure 24H The expression of BrdU in bone marrow from day 3 to day 6 after 5FU treatment is shown. + Cell-to-bone surface ratio. Figure 24I Shown are BrdU near the bone surface and near vascular / adipose structures on days 3 to 6 after 5FU treatment. +The percentage of cells. Figure 24J Representative whole bone sections are shown, demonstrating N-cad-driven tomato proliferation on the bone surface and in the central bone marrow. + cell. Figure 24K Figure 3 shows N-cad-driven tomato expression in central bone marrow (CM) and bone of control N-cad-TdT mice and mice 3 days after 5FU treatment. + Absolute cell numbers. N = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars, sem.

[0100] Figures 25A-25B :N-cad-CreER T Generation of mouse lines. Figure 25A N-cad-CreER is shown T Generation of mouse lines. Figure 25B N-cad-CreER after 3 TMX injections is shown T , Lineage tracing of R26-tdT;Col2.3-GFP mice; blood vessels stained with CD31 and Ve-cadherin antibodies.

[0101] Figures 26A-26E : Sorting strategies and characteristic gene expression of niche cells. Figure 26A Niche cells in the endosteal region harvested from digested osteoblasts are shown. Figure 26B Niche cells from perivascular and sinusoidal regions harvested from digested bone marrow cells are shown. Figure 26C NG2-RFP is shown + Localization of cells in the endosteal and periarterial regions. Figure 26D -E shows heat maps of osteochondrogenic and adipogenic progenitor gene expression in niche cells.

[0102] Figures 27A-27E : Lineage tracing in N-cad-CreERT;R26-tdT mice at early time points after TMX induction. Figure 27A -B shows representative images of trabecular bone (TB) and cortical bone (CB) in N-cad-CreERT;R26-tdT;Col2.3-GFP mice 24 hours and 2 weeks after TMX induction. Scale bar, 20 μm. Figure 27C -E shows representative femoral sections with high-power images of N-cad-CreERT;R26-tdT mice at 6, 14, and 24 hours after TMX induction. Adipocytes were visualized by staining with perilipin antibody.

[0103] Figures 28A-28D:N-cad from early development and injury repair + Characterization of MSCs. Figure 28A Representative images of femoral sections showing N-cad-CreER 24 h after TMX induction at postnatal D2 are shown. T ; N-cad with aggrecan staining in R26-tdT + MSC. Figure 28B Two-month-old N-cad-CreER induced in TMX at E12.5 is shown T Representative femoral sections from R26-tdT mice. Adipocytes were visualized using perilipin antibody staining. Figure 28C Two-day-old N-cad-CreER induced by TMX at E12.5 is shown T Representative femoral sections from R26-tdT mice, stained with an osteopontin antibody to reveal developing osteoblasts. Figure 28D Figure 3 shows the expression of N-cad-CreER induced by TMX in the control group and at 2 weeks after knee cartilage injury at E12.5. T Representative sagittal knee sections in R26-tdT mice. Scale bar, 1 mm.

[0104] Figure 29 : Surviving rHSCs (green, CD150) on day 3 after 5FU treatment were usually detected as single cells adjacent to the bone surface (white, SHG). + Lin ─ CD49b ─ ) images, and proliferating HSCs are often associated with MKs (CD150 + Lin + ) or near blood vessels (red, CD31+).

[0105] Figure 30 : Graph showing in vivo production of human Thpo by 10K ncad+hMSCs and 1000k total hMSCs. Detailed Description of the Invention

[0107] One embodiment of the present invention is a method for expanding a stem cell population obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow. The method comprises regulating N in the stem cell population. 6 -methyladenosine (m 6 A) mRNA modification pathways to expand stem cell numbers.

[0108] Another embodiment of the present invention is a method for expanding a cell population of chimeric antigen receptor (CAR) T cells obtained by modifying T cells obtained from tissue selected from peripheral blood, umbilical cord blood and bone marrow. The method comprises regulating N in the CART cell population.6 -methyladenosine (m 6 A) mRNA modification pathways to expand stem cell numbers.

[0109] In the present invention, the population of stem cells and / or T cells can be obtained from any mammal, such as a human, and from any tissue comprising stem cells and / or progenitor cells and / or T cells. As described above, in a preferred embodiment, the tissue can be peripheral blood, umbilical cord blood or bone marrow.

[0110] As used herein, "expand," "proliferate," and similar terms refer to increasing the number of stem cells and / or CAR T cells in a population in vivo or ex vivo relative to the number of stem cells and / or CAR T cells in the original population using any of the methods disclosed herein. The expansion may be at least 40-fold compared to the original number of stem cells and / or CAR T cells in the population. More preferably, the expansion is at least 2-fold, 4-fold, 5-fold, 8-fold, 10-fold, 15-fold, 20-fold or more compared to the original number of stem cells.

[0111] In the present invention, " stem cell population " refers to a group of substantially undifferentiated cells, which have the ability to generate many different types of cells and have the ability to self-renew. Representative, non-limiting examples of stem cells according to the present invention include bronchial alveolar stem cells (BASC), bulge epithelial stem cells (bESC), corneal epithelial stem cells (CESC), cardiac stem cells (CSC), epidermal neural crest stem cells (eNCSC), embryonic stem cells (ESC), endothelial progenitor cells (EPC), liver oval cells (HOC), hematopoietic stem cells (HSC), hematopoietic stem and progenitor cells (HSPC), keratinocyte stem cells (KSC), mesenchymal stem cells (MSC), neuronal stem cells (NSC), pancreatic stem cells (PSC), retinal stem cells (RSC) and skin-derived precursor cells (SKP).

[0112] For example, hematopoietic stem cells have the ability to self-renew (i.e., expand) and can produce all types of progenitor cells (e.g., CMP, GMP, MEP, and CLP), and ultimately produce all types of blood cells in the hematopoietic system (e.g., erythrocytes, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets). As another example, mesenchymal stem cells are multipotent stromal cells that can differentiate into various cell types (e.g., osteoblasts, chondrocytes, muscle cells, and adipocytes).

[0113] In the present invention, " chimeric antigen receptor (CAR) T cell group " refers to a group of T cells that have been modified with a chimeric antigen receptor capable of binding to a specific antigen such as an antigen on the surface of a cancer cell and may have the ability to target and kill such cancer cells. CAR T cells can be prepared by modifying T cells with chimeric antigen receptors, for example, by introducing DNA encoding a chimeric antigen receptor into T cells to express a chimeric antigen receptor on the surface of T cells (Davila et al. Sci Transl Med 6 (224), 224ra25 (2014); Tasian et al. Ther Adv Hematol 6 (5), 228-241 (2015).

[0114] In the present invention, "modulating", "modulation" and similar terms refer to changes in signal transduction pathways, such as m 6 A protein in the mRNA modification pathway, including but not limited to reducing or increasing the expression level of a protein, altering the sequence of such a protein (by mutation, pre- or post-translational modification, or other means), or inhibiting or activating such a protein (whether by binding, phosphorylation, glycosylation, translocation, or other means). Such modulation can be achieved genetically or pharmacologically.

[0115] In one aspect of the present invention, adjusting m 6 A mRNA modification pathway involves the production of 6 A mutation in a molecule in the mRNA modification pathway is introduced into a stem cell and / or CAR-T cell population. In another aspect of the present invention, m 6 Regulation of A mRNA modification pathways also includes the regulation of stem cells and / or CAR T cells with m 6 A. Contact with a modulator of a molecule in an mRNA pathway. Representative, non-limiting examples of such modulators include small molecules, biologics, antisense RNA, small interfering RNA (siRNA), and combinations thereof.

[0116] In the present invention, the phrase "m 6 A "regulation of molecules in the mRNA modification pathway" refers to changes in m 6 A member of an mRNA modification pathway may have a function that is related to inhibiting or reducing mRNA expression. 6 Non-limiting examples of such "modulation" include increasing or decreasing the activity of a protein involved in the incorporation of N into mRNA. 6 -Methyladenosine modification, removal of N 6 -methyladenosine mRNA modification and / or recognition and processing of N 6For example, regulation may include increasing or decreasing N in mRNA. 6 -methyladenosine modifications, and / or influencing the type and / or distribution of such modifications in mRNA, e.g., by regulating m 6 A writing (such as methyltransferase) and m 6 As another example, regulation can be achieved by regulating the activity of one or more of the A erasers (e.g., demethylases). 6 The activity of a reader (e.g., an RNA-binding protein that recognizes methylated adenosine) is used to increase or decrease the activity of the reader A to recognize N-terminal residues on mRNA. 6 -methyladenosine modification to mediate m 6 A-dependent function of the protein expression or function. Therefore, m 6 A. Modulation of molecules in the mRNA modification pathway regulators can lead to m 6 A. Regulation of the activity and / or expression of molecules upstream or downstream of the mRNA modification process.

[0117] In one aspect, m 6 A. Modulation of an mRNA modification pathway involves the regulation of a molecule selected from the group consisting of: m 6 A mRNA modification reading, m 6 A mRNA modification writing, m 6 A mRNA modification erasers and combinations thereof. 6 Non-limiting examples of A-modifier scripts include those capable of post-transcriptionally installing m 6 A modified methyltransferase, and may include any one selected from METTL3, METTL14, WTAP, KIAA1429, and combinations thereof. 6 Non-limiting examples of A modified erasers include demethylases capable of reversing methylation, and may include any one selected from FTI, ALKBH5, and combinations thereof. 6 A modified read includes the ability to selectively bind m 6 A-methylated mRNA can be used to selectively recognize methylated mRNA to regulate proteins. 6 A modified read may include any one selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, eIF3, and combinations thereof. According to one aspect, m 6A modified reading includes proteins of the YTH domain protein family, including Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, and combinations thereof (see, e.g., Wang et al. Nature, 505(7481): 117-120, 2014; Frayling et al. Science, 316: 889-894, 2007; Zheng et al. Mol. Cell., 49: 18-29, 2012; Cao et al. Open Biol., 6(4): 160003, 2016; Maity et al. The FEBS Journal, 283(9): 1607-1630, 2016).

[0118] As used herein, "introducing mutations" refers to any conventional method for generating changes in the genetic composition of a stem cell population and / or a CAR T cell population. Non-limiting examples of methods for introducing mutations into a stem cell population and / or a CAR T cell population include mutagenesis by ultraviolet irradiation, chemical mutagenesis, targeted mutagenesis such as site-directed mutagenesis of stem cells and / or CAR T cells, and creation of transgenic mice. According to one aspect, mutations can be introduced into stem cells and / or CAR T cells to delete, replace, or modify a gene expressing mRNA. 6 A. Modify a gene or reduce the expression of a molecule in an mRNA modification pathway, such as a molecule selected from the group consisting of: 6 AmRNA modified readings, m 6 A mRNA modification writing, m 6 A mRNA modification erasures and combinations thereof. In one aspect, mutations are introduced to delete, replace, or express mRNA. 6 A mRNA modification reader (e.g., any one selected from Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, eIF3, and combinations thereof) or reduces its expression. In a preferred aspect, a mutation is introduced to delete, replace, or reduce the expression of a gene expressing Ythdf2. In another aspect, a mutation is introduced to delete, replace, or reduce the expression of a gene expressing m 6 A gene that modifies mRNA of a writing object (e.g., any one selected from METTL3, METTL14, WTAP, KIAA1429, and combinations thereof) or reduces its expression. In another aspect, a mutation is introduced to delete, replace, or express a mRNA. 6 A mRNA modifies a gene of an eraser (eg, any one selected from FTI, ALKBH5, and a combination thereof) or reduces its expression.

[0119] In one aspect, stem cells and / or CAR T cells may be exposed to a cell expressing m 6A mutation is introduced by inactivating or deleting at least a portion of the gene for a molecule in the mRNA modification pathway using the Mx1-Cre targeting system (see, for example, Kuhn et al. Science, 269(5229):1427-1429, 1995). In another aspect, short hairpin RNA (shRNA) is introduced into stem cells and / or CAR T cells to reduce the expression of m 6 A mutation in the expression of a gene of a molecule in an mRNA modification pathway. For example, shRNA can be introduced by exposing stem cells and / or CAR T cells to a vector to deliver shRNA, and the vector can be a viral vector such as a lentivirus (see, for example, Chira et al. Oncotarget, 6(31): 30675-30703, 2015). shRNA can be able to trigger gene silencing to regulate gene expression (see, for example, Paddison et al. Genes Dev., 16(8): 948-958, 2002).

[0120] As used herein, “N 6 -methyladenosine mRNA modification pathway regulators (or "m 6 A mRNA modification pathway regulator) is a regulator of m 6 A. Any agent that modulates the activity of any member of an mRNA modification pathway, for example by regulating m 6 A writing (e.g., methyltransferase) and m 6 The activity of one or more of the A erasers (e.g., demethylases) results in, for example, N 6 As another example, the agent may be an agent that increases or decreases the N-methyladenosine modification and / or changes in the type and / or distribution of such modifications in the mRNA. 6 -methyladenosine modification to mediate m 6 A-dependent functions (e.g., by regulating m 6 A is a reagent that reads the activity of a protein (e.g., an RNA binding protein that recognizes methylated adenosine). 6 A mRNA modification pathway regulator can act on mRNA 6 A modified agent, or acting upstream or downstream thereof.

[0121] In one embodiment, by downregulating and / or inhibiting m 6 A member of an mRNA modification pathway, such as downregulating and / or inhibiting m 6 A mRNA modification reader to regulate m 6 A mRNA modification pathway. As used herein, "downregulate" refers to inhibiting or reducing m 6A mRNA modification pathway member or inhibit or reduce its activity. Such downregulation can be achieved by using, for example, antisense RNA, siRNA, antibodies or small molecules. As another example, stem cells and / or CAR T cells can be modified by interacting with mRNA. 6 A mRNA reader is contacted by an inhibitor to downregulate m 6 A mRNA modification readout to inhibit m 6 A mRNA reading pair 6 In one aspect, the downregulated mRNA 6 A mRNA modification reading is selected from Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, eIF3 and combinations thereof. In a preferred aspect, the downregulated m 6 A mRNA modification reader is Ythdf2. m 6Inhibitors of an mRNA modification reader can be selected from: (inhibitors of HNRNPC) hsa-let-7e-5p (MIRT051596), hsa-mir-455-3p (MIRT037890), hsa-mir-30c-5p (MIRT047904), hsa-mir-186-5p (MIRT045150), hsa-mir-744-5p (MIRT037494), hsa-mir-18a-3p (MIRT040851), hsa-mir-484 (MIRT042196), hsa-mir-505-5p (MIRT037959), hsa-mir-615-3p (MIRT039991), hsa-mir-342-3p (MIRT043694), hsa-miR-3607-3p, hsa-miR-30d, hsa-miR-3916, hsa-miR-3162-5p, hsa-miR-1273d, hsa-miR-3161, hsa-miR-30a, hsa-miR-629, hsa-miR-208b, hsa-miR-489, hsa-miR-3148, hsa-miR-2113, hsa-miR-877, hsa-miR-455-5p, hsa-miR-186, hsa-miR-548o, hsa-miR-3139, hsa-miR-320a, hsa-miR-4311, hsa-miR-555, hsa-miR-3605-5p, hsa-miR-515-5p, hsa-miR-144, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-548x, hsa-miR-299-5p, hsa-miR-653, hsa-miR-576-5p, hsa-miR-548p, hsa-miR-586, hsa-miR-888, hsa-miR-3647-3p, hsa-miR-484, hsa-miR-320b, hsa-miR-620, hsa-miR-30b, hsa-miR-548q, hsa-miR-29b-1, hsa-miR-570, hsa-miR-183, hsa-miR-1276, hsa-miR-208a, hsa-miR-186, hsa-miR-28-5p, hsa-miR-330-3p, hsa-miR-548am, hsa-miR-320d, hsa-miR-3175, hsa-miR-3155, hsa-miR-548aa, hsa-miR-519e, hsa-miR-1270,hsa-miR-513b, hsa-miR-599, hsa-miR-518f, hsa-miR-4301, hsa-miR-30c, hsa-miR-3135, hsa-miR-4286, hsa-miR-202, hsa-miR-4263, hsa-miR-4299, hsa-miR-606, hsa-miR-3133, hsa-miR-583, hsa-miR-3125, hsa-miR-501-5p, hsa-miR-7-1, hsa-miR-514b-3p, hsa-miR-3155b, hsa-miR-548d-3p, hsa-miR-224, hsa-miR-7-2, hsa-miR-708, hsa-miR-3199, hsa-miR-514, hsa-miR-30e (see, for example, Helwak et al., Cell, 153(3):654 - 655, 2013; Whisnant et al., MBio 4(2), 2013:e000193); (inhibitors of HNRNPA2B1) hsa-mir-92a-3p (MIRT049721), hsa-mir-30c-5p (MIRT048009), hsa-mir-191-5p (MIRT045809), hsa-let-7f-5p (MIRT051404), hsa-mir-27b-3p (MIRT046213), hsa-mir-877-3p (MIRT037116), hsa-mir-615-3p (MIRT040278), hsa-mir-1260b (MIRT052680), hsa-mir-103a-3p (MIRT027027), hsa-mir-16-5p (MIRT031508), hsa-mir-1296-5p (MIRT036075), hsa-mir-197-3p (MIRT048098), hsa-miR-548j, hsa-miR-3678-3p, hsa-miR-607, hsa-miR-188-5p, hsa-miR-15a, hsa-miR-3653, hsa-miR-371-5p, hsa-miR-550a, hsa-miR-3622b-3p, hsa-miR-548a-5p, hsa-miR-3170, hsa-miR-3148, hsa-miR-556-3p, hsa-miR-490-3p, hsa-miR-559, hsa-miR-200c, hsa-miR-130a, hsa-miR-548y, hsa-miR-548o,hsa-miR-23c,hsa-miR-491-3p,hsa-miR-335,hsa-miR-3667-3p,hsa-miR-466,hsa-miR-23b,hsa-miR-4310,hsa-miR-127-5p,hsa-miR-548b-5p,hsa-miR-616,hsa-miR-16,hsa-miR-338-3p,hsa-miR-3200-5p,hsa-miR-362-3p,hsa-miR-448,hsa-miR-1306,hsa-miR-944,hsa-miR-3684,hsa-miR -373,hsa-miR-103a,hsa-miR-380,hsa-miR-499-5p,hsa-miR-1323,hsa-miR-323-5p,hsa-miR-3674,hsa-miR-1252,hsa-miR-33b,hsa-miR-580,hsa-miR-548c-3p,hsa-miR-103a-2,hsa-miR-548w,hsa-miR-600,hsa-miR-634,hsa-miR-586,hsa-miR-497,hsa-miR-720,hsa-miR-654-3p,hsa-miR- 524-5p,hsa-miR-543,hsa-miR-548q,hsa-let-7f-2,hsa-miR-330-5p,hsa-miR-500a,hsa-miR-548l,hsa-miR-570,hsa-miR-374a,hsa-miR-1184,hsa-miR-649,hsa-miR-424,hsa-miR-3658,hsa-miR-186,hsa-miR-326,hsa-miR-548d-5p,hsa-miR-23a,hsa-miR-15b,hsa-miR-190,hsa-miR-203, hsa-miR-548h,hsa-miR-3136-5p,hsa-miR-618,hsa-miR-551b,hsa-miR-211,hsa-miR-1305,hsa-miR-513b,hsa-miR-96,hsa-miR-2117,hsa-miR-548n,hsa-miR-3910,hsa-miR-217,hsa-miR-892b,hsa-miR-502-5p,hsa-miR-548i,hsa-miR-520d-5p,hsa-miR-4299,hsa-miR-1285,hsa-miR-3133,hsa-miR-483-3p (see, e.g., Hafner et al., Cell, 141(1):129-141, 2010; Helwak et al., Cell, 153(3):654-655, 2013); (inhibitor of Ythdf1) hsa-miR-548g, hsa-miR-204, hsa-miR-3143, hsa-miR-521, hsa-miR-195, hsa-miR-3182, hsa-miR-3941, hsa-miR-34c-3p, hsa-miR-767-3p, hsa-miR-563, hsa-miR-548c-5p, hsa-miR-1911, hsa-miR-26b, hsa-miR-190b, hsa-miR-33a, hsa-miR-329, hsa-miR-221, hsa-miR-612, hsa-miR-3185, hsa-miR-3156-5p, hsa-miR-107, hsa-miR-664, hsa-miR-3657; (inhibitor of Ythdf2) hsa-mir-615-3p (MIRT040054), hsa-mir-106b-5p (MIRT044257), hsa-mir-1 (MIRT023842), miR-145, hsa-miR-3607-3p, hsa-miR-200a, hsa-miR-301a, hsa-miR-519a, hsa-miR-141, hsa-miR-130b, hsa-miR-181b, hsa-miR-301b, hsa-miR-3117-3p, hsa-miR-1236, hsa-miR-181a, hsa-miR-519c-3p, hsa-miR-551b, hsa-miR-519e, hsa-miR-519b-3p, hsa-miR-19b, hsa-miR-1303, hsa-miR-608, hsa-miR-145, hsa-miR-130a, hsa-miR-181c, hsa-miR-323b-3p, hsa-miR-421, hsa-miR-515-5p, hsa-miR-3666, hsa-miR-181d, hsa-miR-146a, hsa-miR-4295, hsa-miR-454, hsa-miR-3919, hsa-miR-19a, hsa-miR-543, hsa-miR-4262 (see, e.g., Helwak et al., Cell, 153(3):654-655, 2013; Selbach et al., Nature, 455(7209):58-63,2008; Yang et al. J Biol Chem., 292(9):3614 - 3623, 2017); (inhibitors of Ythdf3) hsa-miR-582-3p, hsa-miR-579, hsa-miR-520e, hsa-miR-520f, hsa-miR-3152-3p, hsa-miR-106a, hsa-miR-30d, hsa-miR-30a, hsa-miR-93, hsa-miR-508-5p, hsa-miR-29a, hsa-miR-3148, hsa-miR-490-5p, hsa-miR-520b, hsa-miR-20a, hsa-miR-409-3p, hsa-miR-4255, hsa-let-7i, hsa-miR-373, hsa-let-7e, hsa-miR-520c-3p, hsa-miR-3920, hsa-miR-127-5p, hsa-miR-380, hsa-miR-616, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-let-7c, hsa-miR-340, hsa-miR-373, hsa-miR-520a-3p, hsa-miR-144, hsa-miR-1265, hsa-miR-548x, hsa-miR-362-5p, hsa-miR-33b, hsa-miR-26b, hsa-miR-17, hsa-miR-569, hsa-miR-3618, hsa-miR-576-5p, hsa-miR-922, hsa-miR-302a, hsa-miR-106b, hsa-miR-888, hsa-miR-484, hsa-let-7b, hsa-miR-582-5p, hsa-let-7f, hsa-miR-30b, hsa-miR-524-5p, hsa-miR-302d, hsa-let-7d, hsa-miR-513a-5p, hsa-miR-500a, hsa-miR-570, hsa-miR-548l, hsa-miR-105, hsa-miR-374c, hsa-let-7g hsa-miR-372, hsa-miR-3658, hsa-let-7a, hsa-miR-3908, hsa-miR-302b, hsa-miR-526b, hsa-miR-190, hsa-miR-181b, hsa-miR-433, hsa-miR-98, hsa-miR-3606, hsa-miR-595,hsa-miR-548am, hsa-miR-187, hsa-miR-561, hsa-miR-181a, hsa-miR-3155, hsa-miR-655, hsa-miR-302c, hsa-miR-195, hsa-miR-26a, hsa-miR-590-3p, hsa-miR-30c, hsa-miR-502-5p, hsa-miR-495, hsa-miR-137, hsa-miR-181c, hsa-miR-520d-5p, hsa-miR-3942-5p, hsa-miR-202, hsa-miR-302e, hsa-miR-513c, hsa-miR-885-5p, hsa-miR-520a-5p, hsa-miR-583, hsa-miR-1297, hsa-miR-7-1, hsa-miR-520d-3p, hsa-miR-3155b, hsa-miR-3182, hsa-miR-519d, hsa-miR-550a, hsa-miR-7-2, hsa-miR-181d, hsa-miR-190b, hsa-miR-1912, hsa-miR-151-3p, hsa-miR-33a, hsa-miR-525-5p, hsa-miR-20b, hsa-miR-514b-5p, hsa-miR-30e, hsa-miR-4262, hsa-miR-636; (inhibitor of elF3) hsa-mir-92b-3p (MIRT040734), hsa-mir-16-5p (MIRT031705), hsa-mir-18a-3p (MIRT040974), hsa-mir-155-5p (MIRT020771), hsa-mir-484 (MIRT042324), hsa-let-7c-5p (MIRT051776), hsa-miR-3910, hsa-miR-148b, hsa-miR-136, hsa-miR-15a, hsa-miR-488, hsa-miR-500a, hsa-miR-1297, hsa-miR-3159, hsa-miR-374c, hsa-miR-424, hsa-miR-7-1, hsa-miR-186, hsa-miR-195, hsa-miR-15b, hsa-miR-26b, hsa-miR-505, hsa-miR-1206, hsa-miR-653, hsa-miR-1283, hsa-miR-7-2, hsa-miR-196a, hsa-miR-497,hsa-miR-33a, hsa-miR-655, hsa-miR-26a hsa-miR-16, hsa-mir-151a-3p(MIRT043600), hsa-mir-92a-3p(MIRT049064), hsa-mir-615-3p(MIRT039779), hsa-mir-877-3p(MIRT036964), hsa-mir-222-3p(MIRT046746), hsa-mir-423-3p(MIRT042468), hsa-mir-324-3p(MIRT042887), hsa-mir-124-3p(MIRT022932), hsa-miR-3140- 3p,hsa-miR-124,hsa-miR-198,hsa-miR-525-5p,hsa-miR-506,hsa-miR-520a-5p,hsa-miR-196a*hsa-miR-3117-3p,hsa-mir-342-5p(MIRT038210),hsa-mir-378a-5p(MIRT043981),hsa-mir-615-3p(MIRT040086),hsa-let-7b-5p(MIRT052211),hsa-mir-455-3p(MIRT037879),hsa-miR -4267,hsa-miR-590-3p,hsa-mir-106b-5p(MIRT044355),hsa-mir-320a(MIRT044466),hsa-mir-16-5p(MIRT032018),hsa-mir-155-5p(MIRT021009),hsa-miR-4302,hsa-mir-191-5p(MIRT045793),hsa-mir-1303(MIRT035890),hsa-mir-193b-3p(MIRT016316),hsa-mir-222-3p(MIRT04 6640),hsa-mir-532-3p(MIRT037924),hsa-mir-18a-3p(MIRT040929),hsa-mir-92a-3p(MIRT049001),hsa-miR-582-3p,hsa-miR-4265,hsa-miR-218-2,hsa-miR-1271,hsa-miR-340,hsa-miR-221,hsa-miR-20b,hsa-miR-508-3p,hsa-miR-141,hsa-miR-4325,hsa-miR-889,hsa-miR-29a,hsa-miR-129-3p, hsa-miR-129, hsa-miR-96, hsa-miR-3163, hsa-miR-187, hsa-miR-196a, hsa-miR-222, hsa-miR-1179, hsa-miR-182, hsa-miR-9*hsa-miR-32, hsa-miR-143, hsa-miR-4296 (see, e.g., Helwak et al. Cell, 153(3):654 - 656, 2013; Selbach et al. Nature, 455(7209):58 - 63, 2008; Baek et al., Nature, 455(7209):64 - 71, 2008; Leivonen et al. MolCell Proteomics, 10(7), 2011:M110.005322): (inhibitors of YTHDC1) hsa-mir-20a-3p (MIRT038967), hsa-mir-103a-3p (MIRT027037), hsa-mir-1 (MIRT023492), hsa-mir-19b-3p (MIRT031105), hsa-mir-100-5p (MIRT048400), hsa-mir-93-5p (MIRT027989), hsa-mir-16-5p (MIRT031379), hsa-let-7b-5p (MIRT052150), hsa-miR-520f, hsa-miR-300, hsa-miR-15a, hsa-miR-200a, hsa-miR-605, hsa-miR-30d, hsa-miR-30a, hsa-miR-3613-3p, hsa-miR-509-3-5p, hsa-miR-34c-5p, hsa-miR-324-3p, hsa-miR-1248, hsa-miR-152, hsa-miR-548t, hsa-miR-4310, hsa-miR-145, hsa-miR-516a-3p, hsa-miR-16, hsa-miR-3668, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-miR-148b, hsa-miR-509-5p, hsa-miR-103a, hsa-miR-1265, hsa-miR-2115, hsa-miR-548c-3p, hsa-miR-148a, hsa-miR-548p, hsa-miR-513a-3p, hsa-miR-497, hsa-miR-3647-3p, hsa-miR-382,hsa-miR-30b,hsa-miR-543,hsa-let-7f-2,hsa-miR-1269,hsa-miR-3164,hsa-miR-503,hsa-miR-500a,hsa-miR-449a,hsa-miR-141, hsa-miR-424,hsa-miR-3908,hsa-miR-889,hsa-miR-2116,hsa-miR-330-3p,hsa-miR-15b,hsa-miR-181b,hsa-miR-187,hsa-miR-1237 ,hsa-miR-449b,hsa-miR-101,hsa-miR-381,hsa-miR-618,hsa-miR-222,hsa-miR-181a,hsa-miR-432,hsa-miR-96,hsa-miR-19b,hsa -miR-195,hsa-miR-548n,hsa-miR-485-5p,hsa-miR-217,hsa-miR-30c,hsa-miR-495,hsa-miR-137,hsa-miR-1288,hsa-miR-181c,hsa -miR-3942-5p,hsa-miR-548v,hsa-miR-487a,hsa-miR-221,hsa-miR-891b,hsa-miR-205,hsa-miR-195,hsa-miR-4271,hsa-miR-3611 ,hsa-miR-516b,hsa-miR-181d,hsa-miR-154,hsa-miR-646,hsa-miR-153,hsa-miR-34a,hsa-miR-19a,hsa-miR-107,hsa-miR-30e and hsa -miR-4262 (see, e.g., Helwak et al. Cell, 153(3):654-655, 2013; Hafner et al. Cell, 141(1):129-141, 2010; Kishore et al. Nat Methods, 8(7):559-64, 2011; Memczak et al. Nature, 495(7441):333-8, 2013; Selbach et al. Nature, 455(7209):58-63, 2008; Chi et al. Nature. 460(7254):479-86, 2009). ,

[0122] In the present invention, the term "small molecule" includes any chemical or other moiety, other than polypeptides and nucleic acids, that can act to affect biological processes, particularly to modulate m 6A member of an mRNA modification pathway. Small molecules can include any number of therapeutic agents currently known and used, or can be synthesized in libraries of such molecules for screening therapeutic agents for biological functions. Small molecules differ from macromolecules in size. The molecular weight of the small molecules of the present invention is generally less than about 5,000 Daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, and most preferably less than about 500 Da.

[0123] Small molecules include, but are not limited to, organic compounds, peptoids and conjugates thereof. As used herein, the term "organic compound" refers to any carbon-based compound except macromolecules such as nucleic acids and polypeptides. In addition to carbon, organic compounds may also include calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements. Organic compounds may be aromatic or aliphatic forms. Non-limiting examples of organic compounds include acetone, alcohol, aniline, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, olefins, esters, ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds, imidazoles, and phenols. Organic compounds used herein also include nitrated organic compounds and halogenated (e.g., chlorinated) organic compounds.

[0124] Preferred small molecules are relatively easy and inexpensive to manufacture, formulate, or otherwise prepare. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules can be tightly combined with macromolecules to form molecules that have biological activity and improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are conducive to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy properties of the chemical entity.

[0125] In general, a polypeptide mimetic ("peptide mimetic") is a molecule that mimics the biological activity of a polypeptide but is not a peptide in chemical nature. Although in certain embodiments, a peptidomimetic is a molecule that does not contain peptide bonds (i.e., amide bonds between amino acids), the term peptidomimetic can include molecules that are not completely peptide in nature, such as pseudopeptides, hemi-peptides, and peptoids.

[0126] As used herein, the term "biologic" refers to a product that is derived from a biological source rather than a chemical process. Non-limiting examples of "biologics" include proteins, conditioned media, and partially purified products from tissues.

[0127] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein. In the present invention, these terms refer to linked sequences of amino acids, which may be natural, synthetic, or modifications or combinations of natural and synthetic. The term includes antibodies, antibody mimetics, domain antibodies, lipocalins, and targeted proteases. The term also includes vaccines containing peptides or peptide fragments intended to generate antibodies against the peptides or peptide fragments.

[0128] As used herein, "antibodies" include antibodies of the IgG, IgM, IgA, IgD, or IgE classes, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single-chain antibodies, diabodies, bispecific antibodies, and bifunctional antibodies. Antibodies can be monoclonal antibodies, polyclonal antibodies, affinity-purified antibodies, or mixtures thereof, having sufficient binding specificity for a desired epitope or sequence derived therefrom. Antibodies can also be chimeric antibodies. Antibodies can be derived by attaching one or more chemical moieties, peptides, or polypeptide moieties known in the art. Antibodies can be conjugated to chemical moieties. Antibodies can be human or humanized antibodies. These and other antibodies are disclosed in U.S. Published Patent Application No. 20070065447.

[0129] Other antibody-like molecules are also within the scope of the present invention. Such antibody-like molecules include, for example, receptor traps (e.g., etanercept), antibody mimetics (e.g., adnectins, fibronectin-based "addressable" therapeutic binding molecules, e.g., from Compound Therapeutics, Inc.), domain antibodies (the smallest functional fragment of a naturally occurring single-domain antibody (e.g., nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr 15; 64(8): 2853-7)).

[0130] Suitable antibody mimics can generally be used as substitutes for the antibodies and antibody fragments described herein. Such antibody mimics can have advantageous properties (e.g., they can be water-soluble, resistant to proteolysis and / or non-immunogenic). For example, peptides comprising a synthetic β-loop structure that mimics the second complementary determining region (CDR) of a monoclonal antibody have been proposed and produced. See, for example, Saragovi et al., Science.Aug.16,1991;253(5021):792-5. Peptide antibody mimics are also generated by using peptide maps to determine "active" antigen recognition residues, molecular modeling, and molecular dynamics trajectory analysis to design peptide mimics comprising antigen contact residues from multiple CDRs. See, for example, Cassett et al., Biochem Biophys Res Commun.Jul.18,2003;307(1):198-205. Further discussion of principles, methods, etc. that may be applied in the context of the present invention is provided in, for example, Fassina, Immunomethods. October 1994; 5(2): 121-9.

[0131] As used herein, "peptide" includes targeted proteases that are capable of, for example, substrate-targeted inhibition of post-translational modifications, such as disclosed in US Patent Application Publication No. 20060275823.

[0132] As used herein, "antisense" molecules include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (RNA or DNA) that is capable of binding to a target mRNA (sense) or DNA (antisense) sequence. For example, the ability to obtain antisense or sense oligonucleotides based on a cDNA sequence encoding a given protein is described in Stein and Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).

[0133] Antisense molecules can be modified or unmodified RNA, DNA or mixed polymer oligonucleotides. These molecules work by specifically binding to matching sequences, thereby inhibiting peptide synthesis by spatial blocking or by activating RNase H enzyme (Wu-Pong, November 1994, BioPharm, 20-33). Antisense molecules can also change the synthesis of protein or be transported from the nucleus to the cytoplasm by interfering RNA processing (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, the combination of single-stranded DNA and RNA can cause the degradation mediated by the nuclease of heteroduplexes (Wu-Pong, the same). The main chain modified DNA chemicals shown so far as substrates for RNase H are phosphorothioates, phosphorodithioates, trifluoroborate and oligonucleotides containing 2'-arabinose and 2'-fluoroarabinose.

[0134] Antisense molecules can be introduced into cells containing target nucleotide sequences by forming conjugates with ligand-binding molecules, as described, for example, in WO 91 / 04753. Suitable ligand-binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand-binding molecules does not substantially interfere with the ability of the ligand-binding molecules to bind to their corresponding molecules or receptors, or blocks the entry of sense or antisense oligonucleotides or their conjugated forms into cells. Alternatively, sense or antisense oligonucleotides can be introduced into cells containing target nucleic acid sequences by forming oligonucleotide-lipid complexes, as described in WO 90 / 10448.

[0135] The term small interfering RNA ("siRNA") refers to a small inhibitory RNA duplex that induces the RNA interference (RNAi) pathway. (Elbashir, SM et al. Nature 411:494-498 (2001); Caplen, NJ et al. Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001); Harborth, J. et al. J Cell Sci. 114:4557-4565 (2001)). These molecules can vary in length (typically 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in their antisense strand. Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' end of the sense and / or antisense strands. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form a hairpin structure containing the duplex region. As used herein, siRNA molecules are not limited to RNA molecules, but also include chemically modified nucleotides and non-nucleotides. siRNA gene targeting can be achieved by transient siRNA transfer into cells (achieved by classical methods such as liposome-mediated transfection, electroporation or microinjection).

[0136] In another aspect of the invention, the number of stem cells and / or CAR T cells is increased by at least 2-fold. Preferably, the number of stem cells and / or CAR T cells is increased by at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, for example at least 5-fold, including at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. Unexpectedly, this level of stem cell and / or CAR T cell expansion is achieved using the methods of the present invention.

[0137] As described above, the method of the present invention can be used to expand any stem cell population. Preferably, the stem cells that can be expanded according to the method of the present invention can be selected from hematopoietic stem cells (HSC), hematopoietic stem and progenitor cells (HSPC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), cardiac stem cells (CSC), neuronal stem cells (NSC) and combinations thereof. According to one aspect, the stem cell is an HSC. According to another aspect, the stem cell is an MSC.

[0138] The method of the present invention may also be able to expand stem cells so that the total colony forming units (CFU) of the expanded cells increases by at least 5 times, such as 8 times, 10 times, or even 15 times or more. In addition, the method may be able to increase the CFU-granulocyte erythrocyte monocyte megakaryocyte (GEMM) colony by at least 3.8 times, such as at least 4 times, 5 times, and even at least 8 times or more.

[0139] Another embodiment of the present invention is a method for ex vivo expansion of a substantially undifferentiated stem cell population. The method comprises regulating N in the undifferentiated stem cell population. 6 -methyladenosine mRNA modification pathway to expand the number of undifferentiated stem cells without significantly differentiating the stem cell population.

[0140] In this embodiment, a stem cell population is "substantially undifferentiated" if a sufficient number of cells in the stem cell population retain the ability to self-renew and can generate a variety of differentiated cell types when transplanted into a recipient (e.g., in the case of an HSC population, the HSC lineage is repopulated upon transplantation (or in the case of an MSC population, the MSC lineage is repopulated upon transplantation)). As used herein, "no significant differentiation" means that the expanded stem cell population has a sufficient number of cells that maintain multi-lineage differentiation potential so that the full range of target stem lineages can be regenerated when the expanded stem cell population is transplanted into a recipient. Thus, for example, in the case of an HSC population, the expanded HSC population is capable of regenerating the entire hematopoietic cell lineage when transplanted into a recipient. In the case of an MSC population, the expanded MSC population is capable of regenerating the entire mesenchymal cell lineage when transplanted into a recipient.

[0141] Another embodiment is a method for ex vivo expansion of a chimeric antigen receptor (CAR) T cell population. The method comprises regulating the N 6 -methyladenosine (m 6 A) mRNA modification pathway to expand the number of CAR T cells.

[0142] Another embodiment of the present invention is a method for ex vivo expansion of a hematopoietic stem cell (HSC) population obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow. The method comprises regulating N in the HSC population. 6 -methyladenosine mRNA modification pathway to expand HSC populations to sufficient numbers for subsequent transplantation into subjects in need thereof while maintaining multi-lineage differentiation potential in the HSC population.

[0143] Another embodiment of the present invention is a method for ex vivo expansion of a population of mesenchymal stem cells (MSCs) obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow. The method comprises regulating N in the MSC population. 6 -methyladenosine mRNA modification pathway to expand MSC populations to sufficient numbers while maintaining multi-lineage differentiation potential in the MSC population, said MSC population being sufficient for subsequent transplantation into a subject in need thereof.

[0144] Another embodiment of the present invention is a method for ex vivo expansion of a chimeric antigen receptor (CAR) T cell population prepared by modifying T cells obtained from a tissue selected from peripheral blood, umbilical cord blood, and bone marrow. The method comprises regulating N in the CAR T cell population. 6 -methyladenosine (m 6 A) mRNA modification pathway to expand CAR T cell populations to sufficient numbers for subsequent transplantation into subjects in need thereof.

[0145] As used herein, "obtaining" from a tissue refers to any conventional method of collecting or segmenting a tissue from a donor. As previously mentioned, tissue can be any tissue comprising stem cells (e.g., HSC and / or MSC) and / or T cells that can be modified by a chimeric antigen receptor. Therefore, for example, tissue can be obtained from a blood sample, such as a peripheral blood or cord blood sample, or from bone marrow. The method for obtaining such samples is well known to those skilled in the art. In the present invention, the sample can be fresh, i.e., obtained from a donor without freezing. In addition, the sample can be further processed to remove excess or unwanted components before amplification. The sample can also be obtained from a stored stock. For example, in the case of peripheral blood or cord blood, a sample can be taken out from a low temperature or otherwise stored library of such blood. Such a sample can be obtained from any suitable donor. Preferably, the donor is a mammal, such as a primate, such as a human. In addition, the sample can be obtained from an autologous or allogeneic donor or source. Preferably, the sample is obtained from an autologous source.

[0146] In this method, "maintaining multi-lineage differentiation potential" refers to the ability of the expanded HSC and / or MSC population to regenerate all types of progenitor cells (e.g., CMPs, GMPs, MEPs, and CLPs) and ultimately all types of blood cells in the hematopoietic system (including, for example, erythrocytes, B lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils, monocytes, macrophages, and platelets) when transplanted into a subject in need of such a transplant.

[0147] In the present invention, the amount of expanded HSC and / or MSC and / or CAR T cells that is "sufficient for subsequent transplantation" generally corresponds to the number of HSC and / or MSC and / or CAR T cells that will result in greater than about 1% engraftment after transplantation. This is an accepted measure of successful transplantation. In the present invention, any conventional method can be used to determine the percentage of engraftment, including the methods described in the examples. This measurement can be performed with or without competitor cells, and is generally and preferably performed without competitor cells. (Zhang, CC, et al., Nat Med, 12(2):240-5, 2006. Zhang, CC and HF Lodish, Blood, 105(11):4314-20, 2005).

[0148] In the above-mentioned in vitro amplification method, N can be achieved as described above. 6 Regulation of the methyladenosine mRNA modification pathway. 6 -methyladenosine mRNA modification pathways may include those that would lead to m 6 A mutation that regulates a molecule in the mRNA modification pathway is introduced into stem cells and / or CAR T cells, or the stem cells and / or CAR T cells are exposed to m 6 A modulator of a molecule in an mRNA modification pathway selected from the group consisting of a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and combinations thereof.

[0149] In one aspect of the ex vivo expansion method, regulating m 6 A mRNA modification pathway involves the regulation of a molecule selected from the group consisting of: m 6 A mRNA modification reading, m 6 A mRNA modification writing, m 6 A mRNA modification erasers and combinations thereof. 6 Non-limiting examples of A-modifier scripts include those capable of post-transcriptionally installing m 6 A modified methyltransferase, and may include any one selected from METTL3, METTL14, WTAP, KIAA1429, and combinations thereof. 6 Non-limiting examples of A modified erasers include demethylases capable of reversing methylation, and may include any one selected from FTI, ALKBH5, and combinations thereof. 6 A modified read includes the ability to selectively bind m 6 A-methylated mRNA can be used to selectively recognize methylated mRNA to regulate proteins. 6A modified read may include any one selected from the group consisting of Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, eIF3, and combinations thereof. According to one aspect, m 6 A modified reading includes proteins of the YTH domain protein family, including Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, and combinations thereof (see, e.g., Wang et al. Nature, 505(7481): 117-120, 2014; Frayling et al. Science, 316: 889-894, 2007; Zheng et al. Mol. Cell., 49: 18-29, 2012; Cao et al. Open Biol., 6(4): 160003, 2016; Maity et al. The FEBS Journal, 283(9): 1607-1630, 2016).

[0150] In one aspect of the ex vivo expansion method, regulating m 6 A mRNA modification pathway involves introducing mutations into stem cells and / or CAR-T cells to delete, replace, or express mRNAs. 6 A mutation that deletes, replaces, or reduces the expression of a gene that expresses a molecule in an mRNA modification pathway. For example, the mutation deletes, replaces, or reduces the expression of a gene that expresses a molecule selected from the group consisting of: 6 A mRNA modification reading, m 6 AmRNA modified writing, m 6 A mRNA erasures and combinations thereof. In one aspect, the mutation deletes, replaces, or expresses mRNA. 6 A mRNA modification reading (e.g., mRNA selected from Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, eIF3, and combinations thereof) 6 In one preferred aspect, the mutation deletes, replaces, or reduces the expression of a gene expressing Ythdf2. In another aspect, the mutation deletes, replaces, or reduces the expression of a gene expressing mRNA. 6 A gene of an mRNA-modified eraser (e.g., selected from the group consisting of FTO, ALKBH5, and combinations thereof) 6 A mRNA modification erases or reduces its expression. In another aspect, the mutation deletes, replaces, or expresses mRNA. 6 A gene of an mRNA modification script (e.g., selected from the group consisting of METTL3, METTL14, WTAP, KIAA1429, and combinations thereof) 6 A mRNA modification script) or reducing its expression.

[0151] In one aspect of the ex vivo expansion method, mutations can be introduced by any of the methods previously disclosed herein. For example, mutations can be introduced by exposing stem cells and / or CAR T cells to an Mx1-Cre targeting system (see, e.g., Kuhn et al., Science, 269(5229):1427-1429, 1995) that causes the expression of m 6 In another aspect, short hairpin RNA (shRNA) is introduced into stem cells and / or CAR T cells to reduce the expression of mRNA. 6 A mutation in the expression of a gene of a molecule in an mRNA modification pathway. For example, shRNA can be introduced by exposing stem cells and / or CAR-T cells to a vector to deliver shRNA, which can be a viral vector such as a slow virus (see, for example, Chira et al. Oncotarget, 6(31): 30675-30703, 2015). shRNA may be able to trigger gene silencing to regulate gene expression (see, for example, Paddison et al. Genes Dev., 16(8): 948-958, 2002).

[0152] In these ex vivo amplification methods, according to one aspect, regulating m 6 A mRNA modification pathway includes downregulation and / or inhibition of m 6 A member of an mRNA modification pathway, such as downregulating and / or inhibiting m 6 A mRNA modification reading. As used herein, "downregulate" refers to inhibiting or reducing m 6 A mRNA modification pathway member or inhibit or reduce its activity. Such downregulation can be achieved by using, for example, antisense RNA, siRNA, antibodies or small molecules. As another example, stem cells and / or CAR T cells can be modified by interacting with mRNA. 6 A mRNA reader is contacted by an inhibitor to downregulate m 6 A mRNA modification readout to inhibit m 6 A mRNA reading pair 6 In one aspect, the downregulated mRNA 6 A mRNA modification reading is selected from Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1, eIF3 and combinations thereof. In a preferred aspect, the downregulated m 6A mRNA modification reader is Ythdf2. The RNA decay role of Ythdf2 has been previously elucidated (see, for example, Wang et al. Nature 505(7481):117-120, 2014). 6Inhibitors of an mRNA modification reader can be selected from: (inhibitors of HNRNPC) hsa-let-7e-5p (MIRT051596), hsa-mir-455-3p (MIRT037890), hsa-mir-30c-5p (MIRT047904), hsa-mir-186-5p (MIRT045150), hsa-mir-744-5p (MIRT037494), hsa-mir-18a-3p (MIRT040851), hsa-mir-484 (MIRT042196), hsa-mir-505-5p (MIRT037959), hsa-mir-615-3p (MIRT039991), hsa-mir-342-3p (MIRT043694), hsa-miR-3607-3p, hsa-miR-30d, hsa-miR-3916, hsa-miR-3162-5p, hsa-miR-1273d, hsa-miR-3161, hsa-miR-30a, hsa-miR-629, hsa-miR-208b, hsa-miR-489, hsa-miR-3148, hsa-miR-2113, hsa-miR-877, hsa-miR-455-5p, hsa-miR-186, hsa-miR-548o, hsa-miR-3139, hsa-miR-320a, hsa-miR-4311, hsa-miR-555, hsa-miR-3605-5p, hsa-miR-515-5p, hsa-miR-144, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-548x, hsa-miR-299-5p, hsa-miR-653, hsa-miR-576-5p, hsa-miR-548p, hsa-miR-586, hsa-miR-888, hsa-miR-3647-3p, hsa-miR-484, hsa-miR-320b, hsa-miR-620, hsa-miR-30b, hsa-miR-548q, hsa-miR-29b-1, hsa-miR-570, hsa-miR-183, hsa-miR-1276, hsa-miR-208a, hsa-miR-186, hsa-miR-28-5p, hsa-miR-330-3p, hsa-miR-548am, hsa-miR-320d, hsa-miR-3175, hsa-miR-3155, hsa-miR-548aa, hsa-miR-519e, hsa-miR-1270,hsa-miR-513b, hsa-miR-599, hsa-miR-518f, hsa-miR-4301, hsa-miR-30c, hsa-miR-3135, hsa-miR-4286, hsa-miR-202, hsa-miR-4263, hsa-miR-4299, hsa-miR-606, hsa-miR-3133, hsa-miR-583, hsa-miR-3125, hsa-miR-501-5p, hsa-miR-7-1, hsa-miR-514b-3p, hsa-miR-3155b, hsa-miR-548d-3p, hsa-miR-224, hsa-miR-7-2, hsa-miR-708, hsa-miR-3199, hsa-miR-514, hsa-miR-30e (see, e.g., Helwak et al. Cell, 153(3):654 - 655, 2013; Whisnant et al., MBio 4(2), 2013:e000193); (inhibitor of HNRNPA2B1) hsa-mir-92a-3p (MIRT049721), hsa-mir-30c-5p (MIRT048009), hsa-mir-191-5p (MIRT045809), hsa-let-7f-5p (MIRT051404), hsa-mir-27b-3p (MIRT046213), hsa-mir-877-3p (MIRT037116), hsa-mir-615-3p (MIRT040278), hsa-mir-1260b (MIRT052680), hsa-mir-103a-3p (MIRT027027), hsa-mir-16-5p (MIRT031508), hsa-mir-1296-5p (MIRT036075), hsa-mir-197-3p (MIRT048098), hsa-miR-548j, hsa-miR-3678-3p, hsa-miR-607, hsa-miR-188-5p, hsa-miR-15a, hsa-miR-3653, hsa-miR-371-5p, hsa-miR-550a, hsa-miR-3622b-3p, hsa-miR-548a-5p, hsa-miR-3170, hsa-miR-3148, hsa-miR-556-3p, hsa-miR-490-3p, hsa-miR-559, hsa-miR-200c, hsa-miR-130a, hsa-miR-548y, hsa-miR-548o,hsa-miR-23c,hsa-miR-491-3p,hsa-miR-335,hsa-miR-3667-3p,hsa-miR-466,hsa-miR-23b,hsa-miR-4310,hsa-miR-127-5p,hsa-miR-548b-5p,hsa-miR-616,hsa-miR-16,hsa-miR-338-3p,hsa-miR-3200-5p,hsa-miR-362-3p,hsa-miR-448,hsa-miR-1306,hsa-miR-944,hsa-miR-3684,hsa-miR -373,hsa-miR-103a,hsa-miR-380,hsa-miR-499-5p,hsa-miR-1323,hsa-miR-323-5p,hsa-miR-3674,hsa-miR-1252,hsa-miR-33b,hsa-miR-580,hsa-miR-548c-3p,hsa-miR-103a-2,hsa-miR-548w,hsa-miR-600,hsa-miR-634,hsa-miR-586,hsa-miR-497,hsa-miR-720,hsa-miR-654-3p,hsa-miR- 524-5p,hsa-miR-543,hsa-miR-548q,hsa-let-7f-2,hsa-miR-330-5p,hsa-miR-500a,hsa-miR-548l,hsa-miR-570,hsa-miR-374a,hsa-miR-1184,hsa-miR-649,hsa-miR-424,hsa-miR-3658,hsa-miR-186,hsa-miR-326,hsa-miR-548d-5p,hsa-miR-23a,hsa-miR-15b,hsa-miR-190,hsa-miR-203, hsa-miR-548h,hsa-miR-3136-5p,hsa-miR-618,hsa-miR-551b,hsa-miR-211,hsa-miR-1305,hsa-miR-513b,hsa-miR-96,hsa-miR-2117,hsa-miR-548n,hsa-miR-3910,hsa-miR-217,hsa-miR-892b,hsa-miR-502-5p,hsa-miR-548i,hsa-miR-520d-5p,hsa-miR-4299,hsa-miR-1285,hsa-miR-3133,hsa-miR-483-3p (see, for example, Hafner et al. Cell, 141(1):129-141, 2010; Helwak et al. Cell, 153(3):654-655, 2013); (inhibitor of Ythdf1) hsa-miR-548g, hsa-miR-204, hsa-miR-3143, hsa-miR-521, hsa-miR-195, hsa-miR-3182, hsa-miR-3941, hsa-miR-34c-3p, hsa-miR-767-3p, hsa-miR-563, hsa-miR-548c-5p, hsa-miR-1911, hsa-miR-26b, hsa-miR-190b, hsa-miR-33a, hsa-miR-329, hsa-miR-221, hsa-miR-612, hsa-miR-3185, hsa-miR-3156-5p, hsa-miR-107, hsa-miR-664, hsa-miR-3657; (inhibitor of Ythdf2) hsa-mir-615-3p (MIRT040054), hsa-mir-106b-5p (MIRT044257), hsa-mir-1 (MIRT023842), miR-145, hsa-miR-3607-3p, hsa-miR-200a, hsa-miR-301a, hsa-miR-519a, hsa-miR-141, hsa-miR-130b, hsa-miR-181b, hsa-miR-301b, hsa-miR-3117-3p, hsa-miR-1236, hsa-miR-181a, hsa-miR-519c-3p, hsa-miR-551b, hsa-miR-519e, hsa-miR-519b-3p, hsa-miR-19b, hsa-miR-1303, hsa-miR-608, hsa-miR-145, hsa-miR-130a, hsa-miR-181c, hsa-miR-323b-3p, hsa-miR-421, hsa-miR-515-5p, hsa-miR-3666, hsa-miR-181d, hsa-miR-146a, hsa-miR-4295, hsa-miR-454, hsa-miR-3919, hsa-miR-19a, hsa-miR-543, hsa-miR-4262 (see, for example, Helwak et al. Cell, 153(3):654-655, 2013; Selbach et al. Nature, 455(7209):58-63,2008; Yang et al. J Biol Chem., 292(9):3614 - 3623, 2017); (inhibitors of Ythdf3) hsa-miR-582-3p, hsa-miR-579, hsa-miR-520e, hsa-miR-520f, hsa-miR-3152-3p, hsa-miR-106a, hsa-miR-30d, hsa-miR-30a, hsa-miR-93, hsa-miR-508-5p, hsa-miR-29a, hsa-miR-3148, hsa-miR-490-5p, hsa-miR-520b, hsa-miR-20a, hsa-miR-409-3p, hsa-miR-4255, hsa-let-7i, hsa-miR-373, hsa-let-7e, hsa-miR-520c-3p, hsa-miR-3920, hsa-miR-127-5p, hsa-miR-380, hsa-miR-616, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-let-7c, hsa-miR-340, hsa-miR-373, hsa-miR-520a-3p, hsa-miR-144, hsa-miR-1265, hsa-miR-548x, hsa-miR-362-5p, hsa-miR-33b, hsa-miR-26b, hsa-miR-17, hsa-miR-569, hsa-miR-3618, hsa-miR-576-5p, hsa-miR-922, hsa-miR-302a, hsa-miR-106b, hsa-miR-888, hsa-miR-484, hsa-let-7b, hsa-miR-582-5p, hsa-let-7f, hsa-miR-30b, hsa-miR-524-5p, hsa-miR-302d, hsa-let-7d, hsa-miR-513a-5p, hsa-miR-500a, hsa-miR-570, hsa-miR-548l, hsa-miR-105, hsa-miR-374c, hsa-let-7g hsa-miR-372, hsa-miR-3658, hsa-let-7a, hsa-miR-3908, hsa-miR-302b, hsa-miR-526b, hsa-miR-190, hsa-miR-181b, hsa-miR-433, hsa-miR-98, hsa-miR-3606, hsa-miR-595,hsa-miR-548am, hsa-miR-187, hsa-miR-561, hsa-miR-181a, hsa-miR-3155, hsa-miR-655, hsa-miR-302c, hsa-miR-195, hsa-miR-26a, hsa-miR-590-3p, hsa-miR-30c, hsa-miR-502-5p, hsa-miR-495, hsa-miR-137, hsa-miR-181c, hsa-miR-520d-5p, hsa-miR-3942-5p, hsa-miR-202, hsa-miR-302e, hsa-miR-513c, hsa-miR-885-5p, hsa-miR-520a-5p, hsa-miR-583, hsa-miR-1297, hsa-miR-7-1, hsa-miR-520d-3p, hsa-miR-3155b, hsa-miR-3182, hsa-miR-519d, hsa-miR-550a, hsa-miR-7-2, hsa-miR-181d, hsa-miR-190b, hsa-miR-1912, hsa-miR-151-3p, hsa-miR-33a, hsa-miR-525-5p, hsa-miR-20b, hsa-miR-514b-5p, hsa-miR-30e, hsa-miR-4262, hsa-miR-636; (inhibitor of elF3) hsa-mir-92b-3p (MIRT040734), hsa-mir-16-5p (MIRT031705), hsa-mir-18a-3p (MIRT040974), hsa-mir-155-5p (MIRT020771), hsa-mir-484 (MIRT042324), hsa-let-7c-5p (MIRT051776), hsa-miR-3910, hsa-miR-148b, hsa-miR-136, hsa-miR-15a, hsa-miR-488, hsa-miR-500a, hsa-miR-1297, hsa-miR-3159, hsa-miR-374c, hsa-miR-424, hsa-miR-7-1, hsa-miR-186, hsa-miR-195, hsa-miR-15b, hsa-miR-26b, hsa-miR-505, hsa-miR-1206, hsa-miR-653, hsa-miR-1283, hsa-miR-7-2, hsa-miR-196a, hsa-miR-497,hsa-miR-33a, hsa-miR-655, hsa-miR-26a hsa-miR-16, hsa-mir-151a-3p(MIRT043600), hsa-mir-92a-3p(MIRT049064), hsa-mir-615-3p(MIRT039779), hsa-mir-877-3p(MIRT036964), hsa-mir-222-3p(MIRT046746), hsa-mir-423-3p(MIRT042468), hsa-mir-324-3p(MIRT042887), hsa-mir-124-3p(MIRT022932), hsa-miR-3140- 3p,hsa-miR-124,hsa-miR-198,hsa-miR-525-5p,hsa-miR-506,hsa-miR-520a-5p,hsa-miR-196a*hsa-miR-3117-3p,hsa-mir-342-5p(MIRT038210),hsa-mir-378a-5p(MIRT043981),hsa-mir-615-3p(MIRT040086),hsa-let-7b-5p(MIRT052211),hsa-mir-455-3p(MIRT037879),hsa-miR -4267,hsa-miR-590-3p,hsa-mir-106b-5p(MIRT044355),hsa-mir-320a(MIRT044466),hsa-mir-16-5p(MIRT032018),hsa-mir-155-5p(MIRT021009),hsa-miR-4302,hsa-mir-191-5p(MIRT045793),hsa-mir-1303(MIRT035890),hsa-mir-193b-3p(MIRT016316),hsa-mir-222-3p(MIRT04 6640),hsa-mir-532-3p(MIRT037924),hsa-mir-18a-3p(MIRT040929),hsa-mir-92a-3p(MIRT049001),hsa-miR-582-3p,hsa-miR-4265,hsa-miR-218-2,hsa-miR-1271,hsa-miR-340,hsa-miR-221,hsa-miR-20b,hsa-miR-508-3p,hsa-miR-141,hsa-miR-4325,hsa-miR-889,hsa-miR-29a,hsa-miR-129-3p, hsa-miR-129, hsa-miR-96, hsa-miR-3163, hsa-miR-187, hsa-miR-196a, hsa-miR-222, hsa-miR-1179, hsa-miR-182, hsa-miR-9*hsa-miR-32, hsa-miR-143, hsa-miR-4296 (see, for example, Helwak et al. Cell, 153(3):654 - 656, 2013; Selbach et al. Nature, 455(7209):58 - 63, 2008; Baek et al., Nature, 455(7209):64 - 71, 2008; Leivonen et al. Mol Cell Proteomics, 10(7), 2011:M110.005322): (inhibitors of YTHDC1) hsa-mir-20a-3p (MIRT038967), hsa-mir-103a-3p (MIRT027037), hsa-mir-1 (MIRT023492), hsa-mir-19b-3p (MIRT031105), hsa-mir-100-5p (MIRT048400), hsa-mir-93-5p (MIRT027989), hsa-mir-16-5p (MIRT031379), hsa-let-7b-5p (MIRT052150), hsa-miR-520f, hsa-miR-300, hsa-miR-15a, hsa-miR-200a, hsa-miR-605, hsa-miR-30d, hsa-miR-30a, hsa-miR-3613-3p, hsa-miR-509-3-5p, hsa-miR-34c-5p, hsa-miR-324-3p, hsa-miR-1248, hsa-miR-152, hsa-miR-548t, hsa-miR-4310, hsa-miR-145, hsa-miR-516a-3p, hsa-miR-16, hsa-miR-3668, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-miR-148b, hsa-miR-509-5p, hsa-miR-103a, hsa-miR-1265, hsa-miR-2115, hsa-miR-548c-3p, hsa-miR-148a, hsa-miR-548p, hsa-miR-513a-3p, hsa-miR-497, hsa-miR-3647-3p, hsa-miR-382,hsa-miR-30b, hsa-miR-543, hsa-let-7f-2, hsa-miR-1269, hsa-miR-3164, hsa-miR-503, hsa-miR-500a, hsa-miR-449a, hsa-miR-141, hsa-miR-424, hsa-miR-3908, hsa-miR-889, hsa-miR-2116, hsa-miR-330-3p, hsa-miR-15b, hsa-miR-181b, hsa-miR-187, hsa-miR-1237, hsa-miR-449b, hsa-miR-101, hsa-miR-381, hsa-miR-618, hsa-miR-222, hsa-miR-181a, hsa-miR-432, hsa-miR-96, hsa-miR-19b, hsa-miR-195, hsa-miR-548n, hsa-miR-485-5p, hsa-miR-217, hsa-miR-30c, hsa-miR-495, hsa-miR-137, hsa-miR-1288, hsa-miR-181c, hsa-miR-3942-5p, hsa-miR-548v, hsa-miR-487a, hsa-miR-221, hsa-miR-891b, hsa-miR-205, hsa-miR-195, hsa-miR-4271, hsa-miR-3611, hsa-miR-516b, hsa-miR-181d, hsa-miR-154, hsa-miR-646, hsa-miR-153, hsa-miR-34a, hsa-miR-19a, hsa-miR-107, hsa-miR-30e and hsa-miR-4262 (see, e.g., Helwak et al., Cell, 153(3):654-655, 2013; Hafner et al., Cell, 141(1):129-141, 2010; Kishore et al., Nat Methods, 8(7):559-64, 2011; Memczak et al., Nature, 495(7441):333-8, 2013; Selbach et al., Nature, 455(7209):58-63, 2008; Chi et al., Nature. 460(7254):479-86, 2009).

[0153] In these ex vivo expansion methods, preferably stem cells are selected from HSC, hematopoietic stem and progenitor cells (HSPC), endothelial progenitor cells (EPC), mesenchymal stem cells (MSC), cardiac stem cells (CSC), neuronal stem cells (NSC) and combinations thereof. According to certain aspects, the stem cell is HSC. According to other aspects, the stem cell is MSC. The ex vivo expansion method can also use a cell population comprising CAR T cells. In these methods, HSC and / or MSC are obtained from a mammal such as a primate or human tissue selected from umbilical cord blood, peripheral blood and bone marrow, although any tissue containing HSC and / or MSC can be used.

[0154] In another aspect of the method of expanding a hematopoietic stem cell (HSC) population ex vivo, the expansion of the stem cell number is at least 2-fold, e.g., at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold or at least 20-fold or more.

[0155] In another aspect of the method for ex vivo expansion of a mesenchymal stem cell (MSC) population, the expansion of the stem cell number is at least 2-fold, e.g., at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, or at least 20-fold or more.

[0156] In another aspect of the method for expanding a CAR T cell population in vitro, the expansion of the number of CAR T cells is at least 2-fold, such as at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, including at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold or at least 20-fold or more.

[0157] Another embodiment of the invention is an expanded substantially undifferentiated stem cell population produced by a method of the invention, such as a method for ex vivo expansion of a substantially undifferentiated stem cell population or a method for ex vivo expansion of a hematopoietic stem cell (HSC) population.

[0158] Another embodiment of the present invention is an expanded substantially undifferentiated stem cell population produced by a method of the present invention, such as a method for ex vivo expansion of a substantially undifferentiated stem cell population or a method for ex vivo expansion of a mesenchymal stem cell (MSC) population.

[0159] Another embodiment of the present invention is an expanded CAR T cell population prepared by a method of the present invention, e.g., a method for ex vivo expansion of a CAR T cell population.

[0160] Another embodiment of the present invention is a method for expanding hematopoietic stem cells (HSCs) at least 2-fold ex vivo, wherein the expanded HSCs are capable of reconstituting the HSC lineage upon transplantation into a mammal in need thereof.6 A deletion, replacement, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into stem cells, and the HSC population is cultured in an appropriate culture medium.

[0161] Another embodiment of the present invention is a method for expanding mesenchymal stem cells (MSCs) at least 2-fold ex vivo, wherein the expanded MSCs are capable of reconstituting the MSC lineage upon transplantation into a mammal in need thereof. 6 A deletion, replacement, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into stem cells, and the HSC population is cultured in an appropriate culture medium.

[0162] Another embodiment of the present invention is a method for expanding chimeric antigen receptor (CAR) T cells ex vivo by at least 2-fold, wherein the expanded CAR T cells are capable of treating cancer and / or blood disorders when transplanted into a mammalian subject in need thereof. The method comprises: 6 A deletion, substitution, or expression-reducing mutation of a gene for an mRNA modification reader is introduced into CAR T cells, and the CAR T cell population is cultured in an appropriate culture medium.

[0163] In this aspect of the invention, "capable of reconstructing the HSC lineage" refers to the fact that the amplified HSC, when transplanted into a suitable mammalian subject, results in a transplantation greater than 1% in the recipient, and the transplanted cells are capable of differentiating into the cell lineages necessary for a normal functioning hematopoietic system. In this aspect of the invention, "capable of reconstructing the MSC lineage" refers to the fact that the amplified MSC, when transplanted into a suitable mammalian subject, results in a transplantation greater than 1% in the recipient, and the transplanted cells are capable of differentiating into the cell lineages necessary for a normal functioning hematopoietic system. In this aspect of the invention, "capable of treating cancer and / or blood diseases" refers to the fact that the amplified CAR T cells, when transplanted into a suitable mammalian subject, are capable of providing treatment for cancer and / or blood diseases (such as at least one of leukemia and lymphoma) that the mammalian subject is suffering from. In this method, "suitable culture medium", "fluid culture medium" and "culture medium" used interchangeably herein refer to a physiologically balanced salt solution that can maintain a stem cell population and / or CAR T cell population for a desired time, which solution can optionally be supplemented with a suitable m 6A mRNA modification pathway regulator. Such basal culture media are well known in the art. A non-limiting example of a basal culture medium suitable for HSCs is StemSpan Media (Stem Cell Technologies; catalog number 09600), which is supplemented with 10 ug / ml heparin, 5X penicillin / streptomycin, 10 ng / ml recombinant mouse (rm) stem cell factor, and 20 ng / ml rm-thrombopoietin.

[0164] In one aspect of the invention, the ex vivo expansion of HSCs and / or MSCs and / or CAR T cells by at least 2-fold can be performed by any of the methods described herein. For example, the method can involve introducing deletions, replacements, or expression of m 6 A mutation that modifies the mRNA reading, such as a gene for Ythdf2, or reduces its expression. In addition, mutations can be introduced by any of the methods described herein, such as by exposing stem cells and / or CAR T cells to an agent that modifies the expression of mRNA. 6 A mRNA modification reader is an Mx1-Cre targeting system that inactivates or deletes at least a portion of a gene. Mutations can also be introduced by incorporating shRNA into stem cells and / or CAR-T cells to reduce the expression of m 6 A mRNA modification reader of the gene expression. Also provided is the introduction of other mRNAs targeting the herein described 6 A. Other methods for mutation of mRNA modification reads.

[0165] In one aspect of this embodiment, HSC and / or MSC and / or CAR T cells are obtained from a mammalian tissue selected from umbilical cord blood, peripheral blood and bone marrow, preferably a primate or human tissue. In this embodiment, the number of HSC and / or MSC and / or CART cells is increased by at least 2 times, for example, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, including at least 5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times or more.

[0166] Another embodiment of the present invention is a kit for expanding a population of hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), and / or CAR T cells for subsequent transplantation into a subject in need thereof. The kit comprises a kit for causing expression of m 6A system for introducing a deletion, replacement or reduced expression mutation of a gene that modifies an mRNA reader into an HSC, MSC and / or CAR T cell population, and instructions for use thereof. Preferably, in the kit, the system for introducing a mutation into an HSC, MSC and / or CAR-T cell population includes one or more reagents capable of introducing a deletion, replacement or reduced expression mutation of a gene that expresses Ythdf2 into an HSC, MSC and / or CAR-T cell population. For example, the kit may include a system for introducing a mutation into an HSC, MSC and / or CAR-T cell population, the system including causing an expression of mRNA to be expressed. 6 A Mx1-Cre targeting system for inactivating or deleting at least a portion of a gene that modifies an mRNA reader. The kit may also include a system for introducing a mutation into a HSC, MSC, and / or CAR T cell population, the system comprising a method for delivering shRNA incorporated into a HSC, MSC, and / or CAR T cell population to reduce the expression of m 6 A reagent for a lentiviral vector that modifies the expression of a gene of an mRNA modified reader. The kit may further comprise a reagent for introducing mutations to modulate mRNA as described herein. 6 A mRNA modification pathway (e.g., by deletion, replacement, expression of m 6 A. Other systems / methods for modifying mRNA of a gene including Ythdf2 or reducing its expression. The kit and components therein can be packaged in any suitable manner for distribution and / or storage.

[0167] In another embodiment, a kit for expanding a hematopoietic stem cell population (HSC) and / or a mesenchymal stem cell population (MSC) and / or a CAR-T cell population for subsequent transplantation into a subject in need thereof is provided. The kit comprises m 6 A. Inhibitors of mRNA modification reads and instructions for use thereof, wherein the inhibitor can be any inhibitor disclosed herein, such as an inhibitor of Ythdf2.

[0168] In one aspect of this embodiment, the kit is capable of expanding the number of stem cells and / or CAR T cells by at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more.

[0169] Another embodiment of the present invention is a method of administering hematopoietic stem cells (HSCs) to a subject in need thereof. The method comprises: (a) administering a gene that results in expression of m 6A method of introducing a deletion, substitution, or reduced expression mutation of a gene that modifies mRNA readings into a sample containing a population of HSCs; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the HSCs to the subject. In this embodiment, the method of introducing a mutation and the method of targeting m 6 A mutation that modifies the mRNA reading is as previously disclosed, for example, a mutation that results in deletion of a gene expressing Ythdf2 or a mutation that results in the incorporation of a shRNA that reduces the expression of a gene expressing Ythdf2 into a HSC population. Furthermore, HSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0170] Another embodiment of the present invention is a method of administering mesenchymal stem cells (MSCs) to a subject in need thereof. The method comprises: (a) administering a cell line that causes the expression of m 6 A method of introducing a deletion, substitution, or reduced expression mutation of a gene of a modified mRNA reader into a sample containing a population of MSCs; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the MSCs to the subject. In this embodiment, the method of introducing a mutation and the method of targeting mRNA modification are 6 A mutation that modifies the mRNA reading is as previously disclosed, for example, a mutation that results in deletion of a gene expressing Ythdf2 or a mutation that results in the incorporation of a shRNA that reduces the expression of a gene expressing Ythdf2 into a MSC population. Furthermore, MSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0171] Another embodiment of the present invention is a method of administering a chimeric antigen receptor (CAR) T cell to a subject in need thereof. The method comprises: (a) causing expression of m 6 A. introducing a deletion, replacement, or reduced expression mutation of a gene of a modified mRNA reader into a sample containing a population of CAR T cells; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; and (c) administering the CAR T cells to the subject. In this embodiment, the method of introducing a mutation and the method of targeting m 6A mutation in the mRNA modification reading is as previously disclosed, for example, a mutation that results in deletion of a gene expressing Ythdf2 or a mutation that results in the incorporation of an shRNA that reduces the expression of a gene expressing Ythdf2 into a CAR T cell population. In addition, the CAR T cells can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0172] Another embodiment of the present invention is a method of administering hematopoietic stem cells (HSCs) to a subject in need thereof. The method comprises: (a) in a suitable culture medium, 6 A. culturing a sample containing a population of HSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 (A) an inhibitor of mRNA modification readings; and (c) administering HSCs to a subject. In this method, the inhibitor is as previously disclosed, for example, an inhibitor of Ythdf2. In addition, the HSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, for example, a human.

[0173] Another embodiment of the present invention is a method of administering mesenchymal stem cells (MSCs) to a subject in need thereof. The method comprises: (a) in a suitable culture medium, 6 A. culturing a sample containing a population of MSCs in the presence of an inhibitor of mRNA modification for a sufficient period of time to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 (A) an inhibitor of mRNA modification readings; and (c) administering MSCs to the subject. In this method, the inhibitor is as previously disclosed, for example, an inhibitor of Ythdf2. In addition, the MSCs can be obtained from any suitable tissue, such as a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0174] Another embodiment of the present invention is a method of administering chimeric antigen receptor (CAR) T cells to a subject in need thereof. The method comprises: (a) in a suitable culture medium, in the presence of 6 A. culturing a sample containing a population of CAR T cells in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6A mRNA modification reading inhibitor; and (c) administering CAR T cells to the subject. In this method, the inhibitor is as previously disclosed, for example, an inhibitor of Ythdf2. In addition, the CAR T cells can be obtained from any suitable tissue, such as a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0175] Another embodiment of the present invention is a method for reconstructing bone marrow in a subject in need thereof. The method comprises: (a) causing expression of m 6 A method of introducing a deletion, substitution, or reduced expression mutation of a gene that modifies mRNA readings into a sample containing a population of HSCs; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the HSCs to the subject. In this embodiment, the method of introducing a mutation and the method of targeting m 6 A mutation that modifies the mRNA reading is as previously disclosed, for example, a mutation that results in deletion of a gene expressing Ythdf2 or a mutation that results in the incorporation of a shRNA that reduces the expression of a gene expressing Ythdf2 into a HSC population. Furthermore, HSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0176] Another embodiment of the present invention is a method for reconstructing bone marrow in a subject in need thereof. The method comprises: (a) causing expression of m 6 A method of introducing a deletion, substitution, or reduced expression mutation of a gene of a modified mRNA reader into a sample containing a population of MSCs; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; and (c) administering the MSCs to the subject. In this embodiment, the method of introducing a mutation and the method of targeting mRNA modification are 6 A mutation that modifies the mRNA reading is as previously disclosed, for example, a mutation that results in deletion of a gene expressing Ythdf2 or a mutation that results in the incorporation of a shRNA that reduces the expression of a gene expressing Ythdf2 into a MSC population. Furthermore, MSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0177] Another embodiment of the present invention is a method for treating cancer and / or blood disorders in a subject in need thereof. The method comprises: (a) causing expression of m 6A. introducing a deletion, replacement, or reduced expression mutation of a gene of a modified mRNA reader into a sample containing a population of CAR T cells; (b) culturing the sample in a suitable culture medium for a sufficient period of time to expand the number of CAR T cells in the sample to a number sufficient for transplantation into a subject; and (c) administering the CAR T cells to the subject. In this embodiment, the method of introducing a mutation and the method of targeting m 6 A mutation in the mRNA modification reading is as previously disclosed, for example, a mutation that results in deletion of a gene expressing Ythdf2 or a mutation that results in the incorporation of an shRNA that reduces the expression of a gene expressing Ythdf2 into a CAR T cell population. In addition, the CAR T cells can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, such as a human.

[0178] In yet another embodiment, a method for reconstituting bone marrow in a subject in need thereof is provided. The method comprises (a) in a suitable culture medium, 6 A. culturing a sample containing a population of HSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of HSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 (A) an inhibitor of mRNA modification readings; and (c) administering HSCs to a subject. In this method, the inhibitor is as previously disclosed, for example, an inhibitor of Ythdf2. In addition, the HSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, for example, a human.

[0179] In another embodiment, another method of reconstituting bone marrow in a subject in need thereof is provided. The method comprises (a) in a suitable culture medium, 6 A. culturing a sample containing a population of MSCs in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture; 6 (A) an inhibitor of mRNA modification readings; and (c) administering MSCs to the subject. In this method, the inhibitor is as previously disclosed, for example, an inhibitor of Ythdf2. In addition, the MSCs can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow, and the subject can be a mammal, for example, a human.

[0180] In these methods, "reconstruction of bone marrow" refers to the restoration of all or part of the bone marrow in a subject suffering from a disease in which normal bone marrow function has been impaired. Non-limiting examples of such diseases include blood diseases, such as aplastic anemia, myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH) and blood cancers, such as leukemia. Therefore, as used herein, "reconstruction" refers to the ability of transplanted HSC and / or MSC to be successfully implanted in the host and differentiate into all cell lineages typically found in the bone marrow or derived from the bone marrow. Aspects of the methods herein may involve transplanting HSC and / or MSC obtained from a tissue selected from umbilical cord blood, peripheral blood, and bone marrow to a subject to treat blood diseases, such as leukemia and lymphoma.

[0181] In yet another embodiment, another method for treating cancer and / or blood disorders in a subject in need thereof is provided. The method comprises (a) in a suitable culture medium, 6 A. culturing a sample containing a population of chimeric antigen receptor (CAR) T cells in the presence of an inhibitor of mRNA modification readings for a sufficient period of time to allow the number of CAR T cells in the sample to expand to a number sufficient for transplantation into a subject; (b) removing mRNA from the culture. 6 A mRNA modification reader inhibitor; and (c) administering CAR T cells to the subject. In this method, the inhibitor is as previously disclosed, for example, an inhibitor of Ythdf2. In addition, the T cells modified to prepare CAR T cells can be obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood and bone marrow, and the subject can be a mammal, such as a human.

[0182] In these methods, "treating a cancer and / or blood disorder in a subject" refers to eradicating cancer cells, alleviating symptoms, or otherwise reducing the disease state in a subject suffering from a cancer and / or blood disorder. Non-limiting examples of cancer and / or blood disorders include blood disorders such as aplastic anemia, myelodysplastic syndrome (MDS), paroxysmal nocturnal hemoglobinuria (PNH), and blood cancers such as leukemias and lymphomas.

[0183] In these methods, "a time long enough to expand the number of HSCs" refers to the minimum amount of time that the HSCs in the culture are expanded to the number of HSCs sufficient for one or more transplants, and "a time long enough to expand the number of MSCs" refers to the minimum amount of time that the MSCs in the culture are expanded to the number of MSCs sufficient for one or more transplants. Similarly, "a time long enough to expand the number of CAR T cells" refers to the minimum amount of time that the CAR T cells in the culture are expanded to the number of CAR T cells sufficient for one or more transplants. Typically, such a time period can be cultured for at least about 10 days. In some cases, it may be desirable to expand a stem cell and / or CAR T cell (e.g., HSC and / or MSC) population beyond the number required for a single transplant. For example, it may be desirable to expand a stem cell and / or CAR T cell (e.g., HSC and / or MSC) population to a number sufficient for multiple transplants (e.g., about 2 to about 100 transplants). In these cases, the excess cells can be preserved for later use by any conventional method, such as by cryopreservation.

[0184] As previously described, "amount sufficient for transplantation" refers to the minimum number of stem cells, such as HSCs and / or MSCs and / or CAR T cells, necessary to achieve greater than 1% engraftment in the recipient. "Administering HSCs to a subject" refers to conventional methods for delivering HSCs to a subject, including but not limited to surgical and / or intravenous delivery of HSCs. "Administering MSCs to a subject" refers to conventional methods for delivering MSCs to a subject, including but not limited to surgical and / or intravenous delivery of MSCs. "Administering CAR T cells to a subject" refers to conventional methods for delivering CAR T cells to a subject, including but not limited to surgical and / or intravenous delivery of MSCs. In these embodiments, the tissue from which the HSCs and / or MSCs and / or T cells are obtained and the m 6 A. Inhibitors of mRNA modification readings are as disclosed above.

[0185] Another embodiment of the present invention is a method for expanding a hematopoietic stem cell (HSC) population. The method includes culturing the HSC population under conditions sufficient to cause the HSC population to expand by at least 2-fold, wherein the expanded HSC population is suitable for transplantation into a mammal in need thereof. In this embodiment, "conditions sufficient to cause the HSC population to expand" are those that can cause the HSC in the culture to expand, for example, by at least 2-fold, such as at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 8-fold, at least 10-fold, at least 15-fold, at least 20-fold or more. "Suitable for transplantation into a mammal" means that the quantity and quality of the HSC are sufficient to support greater than 1% engraftment in a mammalian recipient in need thereof (e.g., a primate recipient, including a human recipient).

[0186] Another embodiment of the present invention is a method for expanding a group of mesenchymal stem cells (MSCs). The method includes culturing a MSC group under conditions sufficient to cause the MSC group to expand by at least 2 times, wherein the expanded MSC group is suitable for transplantation into a mammal in need thereof. In this embodiment, "conditions sufficient to cause the MSC group to expand" are those conditions that can cause the MSC in the culture to expand, for example, by at least 2 times, for example, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times or more. "Suitable for transplantation into a mammal" means that the quantity and quality of the MSCs are sufficient to support an implantation greater than 1% in a mammalian recipient (e.g., a primate recipient, including a human recipient) in need thereof.

[0187] Another embodiment of the present invention is a method for expanding a chimeric antigen receptor (CAR) T cell population. The method includes culturing a CAR T cell population under conditions sufficient to cause the CAR T cell population to expand by at least 2 times, wherein the expanded CAR T cell population is suitable for transplantation into a mammal in need thereof. In this embodiment, "conditions sufficient to cause the CAR T cell population to expand" are those conditions that can cause the CAR T cells in the culture to expand, for example, by at least 2 times, for example, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 5 times, at least 8 times, at least 10 times, at least 15 times, at least 20 times or more. "Suitable for transplantation into a mammal" refers to a quantity and quality of CAR T cells sufficient to support greater than 1% implantation in a mammalian receptor (e.g., a primate receptor, including a human receptor) in need thereof.

[0188] Another embodiment of the present invention is a method for treating a subject in need of a bone marrow transplant, a peripheral blood transplant, or an umbilical cord blood transplant, the method comprising administering to the subject a population of hematopoietic stem cells (HSCs) obtained by the methods disclosed herein, particularly a method for expanding a population of HSCs. The subject can be a mammal, such as a human.

[0189] Another embodiment of the present invention is a method for treating a subject in need of a bone marrow transplant, a peripheral blood transplant, or an umbilical cord blood transplant, the method comprising administering to the subject a population of mesenchymal stem cells (MSCs) obtained by the methods disclosed herein, particularly a method for expanding a population of mesenchymal stem cells (MSCs). The subject can be a mammal, such as a human.

[0190] Another embodiment of the present invention is a method for treating a subject with cancer and / or a blood disorder, the method comprising administering to the subject a CAR T cell population obtained by the methods disclosed herein (particularly a method for expanding a CAR T cell population). The subject can be a mammal, such as a human.

[0191] Another embodiment of the present invention is a method for expanding a hematopoietic stem cell (HSC) group. The method comprises: (a) obtaining a tissue sample comprising an HSC group from a mammal; (b) amplifying the HSC group from the sample in vitro, wherein (i) the HSC group is expanded at least 2 times; and (ii) the total colony-forming units of the expanded HSC group are increased by at least 5 times. In one aspect of this embodiment, the HSC group is expanded at least 4 times, such as at least 5 times, including at least 8 times, at least 10 times, at least 15 times, at least 20 times or more. In another aspect of this embodiment, the mammal is a primate, including a human. Preferably, the human needs a peripheral blood transplant, an umbilical cord blood transplant or a bone marrow transplant. On the other hand, the tissue sample is obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood and bone marrow.

[0192] Another embodiment of the present invention is a method for expanding a mesenchymal stem cell (MSC) group. The method includes (a) obtaining a tissue sample comprising an MSC group from a mammal; (b) amplifying the MSC group from the sample in vitro, wherein (i) the MSC group is expanded at least 2 times. In one aspect of this embodiment, the MSC group is expanded at least 2.5 times, such as at least 3 times, at least 3.5 times, at least 4 times, such as at least 5 times, including at least 8 times, at least 10 times, at least 15 times, at least 20 times or more. In another aspect of this embodiment, the mammal is a primate, including a human. Preferably, the human needs a peripheral blood transplant, an umbilical cord blood transplant or a bone marrow transplant. On the other hand, the tissue sample is obtained from any suitable tissue, such as a tissue selected from umbilical cord blood, peripheral blood and bone marrow.

[0193] Another embodiment of the present invention is a method for expanding a chimeric antigen receptor (CAR) T cell group. The method includes (a) obtaining a tissue sample comprising a T cell group from a mammal; (b) modifying the T cell group with a chimeric antigen receptor to provide a CAR T cell group; (c) amplifying the CAR T cell group from the sample in vitro, wherein (i) the CAR T cell group is expanded at least 2 times. In one aspect of this embodiment, the CAR T cell group is expanded at least 2.5 times, for example, at least 3 times, at least 3.5 times, at least 4 times, for example, at least 5 times, including at least 8 times, at least 10 times, at least 15 times, at least 20 times or more. In another aspect of this embodiment, the mammal is a primate, including a human. Preferably, the human suffers from cancer and / or a blood disease. On the other hand, the tissue sample is obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood and bone marrow.

[0194] Another embodiment of the present invention is a method for reconstructing a hematopoietic stem cell lineage in a recipient in need thereof. The method comprises: (a) obtaining a tissue sample comprising an HSC population from a mammal; (b) amplifying the HSC population from the sample in vitro, wherein: (i) the HSC population is amplified at least 2 times, such as at least 4 times, including at least 8 times, at least 10 times, at least 15 times, at least 20 times or more, and (ii) the total colony-forming units of the amplified HSC population are increased by at least 5 times; and (c) the amplified HSC population is transplanted into a subject in need thereof, such as a mammal, including a primate or a human. In this embodiment, the human recipient requires a peripheral blood transplant, an umbilical cord blood transplant, or a bone marrow transplant. Therefore, on the other hand, the tissue sample is obtained from any suitable tissue, such as a tissue selected from umbilical cord blood, peripheral blood, and bone marrow. The sample can be obtained from an autologous or allogeneic source. Preferably, the sample is obtained from an autologous source.

[0195] Another embodiment of the present invention is a method for reconstructing a mesenchymal stem cell (MSC) lineage in a recipient in need thereof. The method includes (a) obtaining a tissue sample comprising an MSC group from a mammal; (b) amplifying the MSC group from the sample in vitro, wherein: (i) the MSC group is amplified at least 2 times, for example, at least 4 times, including at least 8 times, at least 10 times, at least 15 times, at least 20 times or more; and (c) the amplified MSC group is transplanted into a subject in need thereof, such as a mammal, including a primate or a human. In this embodiment, the human recipient requires a peripheral blood transplant, an umbilical cord blood transplant, or a bone marrow transplant. Therefore, on the other hand, the tissue sample is obtained from any suitable tissue, for example, a tissue selected from umbilical cord blood, peripheral blood, and bone marrow. The sample can be obtained from an autologous or allogeneic source. Preferably, the sample is obtained from an autologous source.

[0196] Another embodiment of the present invention is a method for treating a subject with cancer and / or blood disease. The method includes (a) obtaining a tissue sample comprising a T cell group from a mammal; (b) modifying a T cell group with a chimeric antigen receptor (CAR) to form a CAR T cell group; (c) amplifying the CAR T cell group from the sample in vitro, wherein: (i) the CAR T cell group is expanded at least 2 times, for example, at least 4 times, including at least 8 times, at least 10 times, at least 15 times, at least 20 times or more; and (c) the amplified CAR-T cell group is transplanted into a subject in need thereof, such as a mammal, including primates or people. In this embodiment, the human receptor may suffer from, for example, leukemia and / or lymphoma. Therefore, on the other hand, the tissue sample is obtained from any suitable tissue, for example, selected from umbilical cord blood, peripheral blood and bone marrow tissue. The sample can be obtained from autologous or allogeneic sources. Preferably, the sample is obtained from an autologous source.

[0197] In aspects of the present invention, preferably, the expanded HSC population comprises a cell line selected from the group consisting of CD34 - or CD34 + / CD38 - / low / Thy-1 + / CD90 + / Kit - / lo / Lin - / CD133 + VEGFR2 + (which is a marker of the most primitive and long-term undifferentiated human HSCs); CD150 + / CD48 - / CD244 - (which is a marker for human HSCs and their progenitor cells); and / or CD150 - / CD48 - / CD244 + and CD150 - / CD48 + / CD244 + (which is a marker of non-self-renewing multipotent hematopoietic progenitor cells) and combinations thereof. (See, for example, Mimeault, M., et al., Stem Cells: A Revolution in Therapeutics-Recent Advances in Stem Cell Biology and Their Therapeutic Applications in Regenerative Medicine and Cancer Therapies. Clin Pharmacol Ther., 82(3): 252-64(2007)). In various aspects of the present invention, preferably, the expanded MSC population and / or the MSC population undergoing expansion include MSCs having a phenotype selected from N-Cadherin+ and CD105+ and combinations thereof. That is, the MSC population of any embodiment described herein may include at least one MSC selected from N-Cadherin+MSC and CD105+MSC. In one embodiment, the MSC population includes N-Cadherin+MSC.

[0198] The exact proportion of HSCs and / or MSCs in the population that possess these markers is not critical, as long as the expanded HSC and / or MSC population as a whole is sufficient to result in at least 1% engraftment in the recipient.

[0199] In another embodiment, the present invention is a method for expanding a hematopoietic stem cell population in a mammal in need of such expansion. The method comprises administering to the mammal a therapeutically effective amount of N 6 -methyladenosine (m 6A) Modulators of mRNA modification pathways for a period of time sufficient to allow for at least a 2-fold expansion of the HSC population, resulting in the ability of HSCs to reconstitute hematopoietic lineages in mammals.

[0200] In another embodiment, the present invention is a method for expanding a population of mesenchymal stem cells in a mammal in need of such expansion. The method comprises administering to the mammal a therapeutically effective amount of N 6 -methyladenosine (m 6 A) Modulators of mRNA modification pathways are expressed for a period of time sufficient to allow for at least a 2-fold expansion of the MSC population, resulting in the ability of the MSCs to reconstitute the mesenchymal lineage in mammals.

[0201] In another embodiment, the present invention is a method of expanding a population of chimeric antigen receptor (CAR) T cells in a mammal in need of such expansion. The method comprises administering to the mammal a therapeutically effective amount of N 6 -methyladenosine (m 6 A) A regulator of an mRNA modification pathway for a period of time sufficient to allow the CAR T cell population to expand at least 2-fold, such that the CAR T cells have the ability to treat cancer and / or blood disorders in a mammal.

[0202] In these methods, the modulator can be as previously disclosed herein, and / or the modulation can be performed by any of the methods disclosed herein. For example, the modulator can include a system for introducing a mutation into a population of HSCs and / or MSCs and / or CAR T cells that deletes, replaces, or expresses a gene or proteins that is expressed in a cell or cell line. 6 -methyladenosine (m 6 A) mRNA modification reading material such as Ythdf2 gene or reduce its expression. As another example, the regulator can include N 6 -methyladenosine (m 6 A) Inhibitors of mRNA modification reads, such as inhibitors of Ythdf2. The mammal to be amplified may be a human.

[0203] In another embodiment, the present invention includes a method for isolating mesenchymal stem cells (MSCs) from a biological sample, the method comprising contacting a biological sample having a population of MSCs with one or more N-cadherin antibodies. For example, according to certain aspects, the biological sample comprises a tissue selected from peripheral blood, umbilical cord blood, and bone marrow. In addition, in certain embodiments, the method for isolating MSCs may further comprise isolating the MSCs by regulating the N-cadherin in the MSC population. 6 -methyladenosine (m 6A) mRNA modification pathway (for example, by any method as described herein) is used to increase MSC groups from a biological sample to increase the number of mesenchymal stem cells. In one embodiment, after separation from a biological sample, the MSC groups are increased. In another embodiment, before MSC is isolated from a biological sample, the MSC groups in the biological sample are increased. In one embodiment, the MSC groups are increased to a sufficient number while maintaining the multi-lineage differentiation potential in the MSC groups, which is sufficient for subsequent transplantation into a subject in need thereof, so that the MSC separated by the method can be used for transplantation. For example, the MSC separated can be transplanted into a human subject. According to another embodiment, MSC can be further isolated from a biological sample by contacting with CD105 antibodies (additional to N-cadherin antibodies or as an alternative to N-cadherin antibodies). In another embodiment, by contacting the biological sample with N-cadherin antibodies and detecting those cells bound to N-cadherin antibodies, N-cadherin antibodies can be used to identify the MSC in a biological sample.

[0204] In one embodiment, a method of isolating MSCs comprises isolating 6 A mutation that modulates a molecule in an mRNA modification pathway is introduced into a stem cell or the stem cell is treated with an mRNA selected from a small molecule, a biologic, an antisense RNA, a small interfering RNA (siRNA), and a combination thereof. 6 A. Modulators of molecules in the mRNA modification pathway contact and regulate m 6 A mRNA modification pathway (e.g., by any of the modulation methods described herein) to expand the MSC population. For example, in one embodiment, modulation of m 6 A mRNA modification pathway involves introducing mutations into stem cells to delete, replace, or express mRNAs. 6 A. The mutation deletes, replaces, or reduces the expression of a gene that expresses a molecule in an mRNA modification pathway. In one embodiment, the mutation deletes, replaces, or reduces the expression of a gene that expresses a molecule selected from the group consisting of: m 6 A mRNA modification reading, m 6 A mRNA modification script and m 6 In yet another embodiment, the mutation deletes, replaces, or expresses mRNA. 6 A mRNA modifies the gene of the reader or reduces its expression. In one embodiment, the mutation deletes, replaces the expression of an mRNA selected from the group consisting of 6A mRNA modification reader gene or reduce its expression: Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1 and eIF3. In another embodiment, the mutation deletes, replaces or reduces the expression of the gene expressing Ythdf2. In one embodiment, the mutation deletes, replaces or reduces the expression of mRNA. 6 A mRNA modifies the gene of the eraser or reduces its expression. In one embodiment, the mutation deletes, replaces, or expresses an mRNA selected from FTO and ALKBH5. 6 A mRNA modifies the gene of the eraser or reduces its expression. In another embodiment, the mutation deletes, replaces, or expresses mRNA. 6 A mRNA modifies the gene of the writing object or reduces its expression. In one embodiment, the mutation deletes, replaces the expression of mRNA selected from METTTL3, METTL14, WTAP and KIAA1429 6 A mRNA modifies the gene it writes about or reduces its expression.

[0205] In another embodiment, a method of isolating MSCs comprises exposing the stem cells to a protein expressing m 6 The Mx1-Cre targeting system regulates mRNA modification by inactivating or deleting at least a portion of the gene of a molecule in the mRNA modification pathway. 6 A mRNA modification pathway to expand MSC population. In one embodiment, regulating m 6 A mRNA modification approach involves introducing shRNA into stem cells to reduce the expression of mRNA. 6 A mutation in the expression of a gene of a molecule in the mRNA modification pathway. For example, in one embodiment, shRNA is introduced by exposing stem cells to a lentivirus to deliver the shRNA. According to yet another embodiment, mRNA is modulated. 6 The a mRNA modification pathway includes downregulation of m 6 A mRNA modified reading. In one embodiment, m 6 A mRNA modification reading is selected from Ythdf1, Ythdf2, Ythdf3, Ythdc1, Ythdc2, HNRNPC, HNRNPA2B1 and eIF3. For example, in one embodiment, m 6 A mRNA modification read contains Ythdf2.

[0206] In one embodiment, the method of isolating MSCs further comprises isolating the stem cells by 6 A mRNA modification reader that downregulates mRNA by contact with inhibitors 6A mRNA modification reader is used to expand the MSC population, and the inhibitor is any one described herein, for example, any substance selected from the following: (inhibitors of HNRNPC) hsa-let-7e-5p (MIRT051596), hsa-mir-455-3p (MIRT037890), hsa-mir-30c-5p (MIRT047904), hsa-mir-186-5p (MIRT045150), hsa-mir-744-5p (MIRT037494), hsa-mir-18a-3p (MIRT040851), hsa-mir-484 (MIRT042196), hsa-mir -505-5p(MIRT037959),hsa-mir-615-3p(MIRT039991),hsa-mir-342-3p(MIRT043694),hsa-miR-3607-3p,hsa-miR-30d,hsa-miR-3916,hsa-miR-31 62-5p,hsa-miR-1273d,hsa-miR-3161,hsa-miR-30a,hsa-miR-629,hsa-miR-208b,hsa-miR-489,hsa-miR-3148,hsa-miR-2113,hsa-miR-877,hsa-m iR-455-5p,hsa-miR-186,hsa-miR-548o,hsa-miR-3139,hsa-miR-320a,hsa-miR-4311,hsa-miR-555,hsa-miR-3605-5p,hsa-miR-515-5p,hsa-miR -144,hsa-miR-499-5p,hsa-miR-1323,hsa-miR-548x,hsa-miR-299-5p,hsa-miR-653,hsa-miR-576-5p,hsa-miR-548p,hsa-miR-586,hsa-miR-888, hsa-miR-3647-3p,hsa-miR-484,hsa-miR-320b,hsa-miR-620,hsa-miR-30b,hsa-miR-548q,hsa-miR-29b-1,hsa-miR-570,hsa-miR-183,hsa-miR-1 276,hsa-miR-208a,hsa-miR-186,hsa-miR-28-5p,hsa-miR-330-3p,hsa-miR-548am,hsa-miR-320d,hsa-miR-3175,hsa-miR-3155,hsa-miR-548aa,hsa-miR-519e, hsa-miR-1270, hsa-miR-513b, hsa-miR-599, hsa-miR-518f, hsa-miR-4301, hsa-miR-30c, hsa-miR-3135, hsa-miR-4286, hsa-miR-202, hsa-miR-4263, hsa-miR-4299, hsa-miR-606, hsa-miR-3133, hsa-miR-583, hsa-miR-3125, hsa-miR-501-5p, hsa-miR-7-1, hsa-miR-514b-3p, hsa-miR-3155b, hsa-miR-548d-3p, hsa-miR-224, hsa-miR-7-2, hsa-miR-708, hsa-miR-3199, hsa-miR-514, hsa-miR-30e; (inhibitor of HNRNPA2B1) hsa-mir-92a-3p (MIRT049721), hsa-mir-30c-5p (MIRT048009), hsa-mir-191-5p (MIRT045809), hsa-let-7f-5p (MIRT051404), hsa-mir-27b-3p (MIRT046213), hsa-mir-877-3p (MIRT037116), hsa-mir-615-3p (MIRT040278), hsa-mir-1260b (MIRT052680), hsa-mir-103a-3p (MIRT027027), hsa-mir-16-5p (MIRT031508), hsa-mir-1296-5p (MIRT036075), hsa-mir-197-3p (MIRT048098), hsa-miR-548j, hsa-miR-3678-3p, hsa-miR-607, hsa-miR-188-5p, hsa-miR-15a, hsa-miR-3653, hsa-miR-371-5p, hsa-miR-550a, hsa-miR-3622b-3p, hsa-miR-548a-5p, hsa-miR-3170, hsa-miR-3148, hsa-miR-556-3p, hsa-miR-490-3p, hsa-miR-559, hsa-miR-200c, hsa-miR-130a, hsa-miR-548y, hsa-miR-548o, hsa-miR-23c, hsa-miR-491-3p, hsa-miR-335,hsa-miR-3667-3p, hsa-miR-466, hsa-miR-23b, hsa-miR-4310, hsa-miR-127-5p, hsa-miR-548b-5p, hsa-miR-616, hsa-miR-16, hsa-miR-338-3p, hsa-miR-3200-5p, hsa-miR-362-3p, hsa-miR-448, hsa-miR-1306, hsa-miR-944, hsa-miR-3684, hsa-miR-373, hsa-miR-103a, hsa-miR-380, hsa-miR-499-5p, hsa-miR-1323, hsa-miR-323-5p, hsa-miR-3674, hsa-miR-1252, hsa-miR-33b, hsa-miR-580, hsa-miR-548c-3p, hsa-miR-103a-2, hsa-miR-548w, hsa-miR-600, hsa-miR-634, hsa-miR-586, hsa-miR-497, hsa-miR-720, hsa-miR-654-3p, hsa-miR-524-5p, hsa-miR-543, hsa-miR-548q, hsa-let-7f-2, hsa-miR-330-5p, hsa-miR-500a, hsa-miR-548l, hsa-miR-570, hsa-miR-374a, hsa-miR-1184, hsa-miR-649, hsa-miR-424, hsa-miR-3658, hsa-miR-186, hsa-miR-326, hsa-miR-548d-5p, hsa-miR-23a, hsa-miR-15b, hsa-miR-190, hsa-miR-203, hsa-miR-548h, hsa-miR-3136-5p, hsa-miR-618, hsa-miR-551b, hsa-miR-211, hsa-miR-1305, hsa-miR-513b, hsa-miR-96, hsa-miR-2117, hsa-miR-548n, hsa-miR-3910, hsa-miR-217, hsa-miR-892b, hsa-miR-502-5p, hsa-miR-548i, hsa-miR-520d-5p, hsa-miR-4299, hsa-miR-1285, hsa-miR-3133, hsa-miR-483-3p; (inhibitor of Ythdf1) hsa-miR-548ghsa-miR-204, hsa-miR-3143, hsa-miR-521, hsa-miR-195, hsa-miR-3182, hsa-miR-3941, hsa-miR-34c-3p, hsa-miR-767-3p, hsa-miR-563, hsa-miR-548c-5p, hsa-miR-1911, hsa-miR-26b, hsa-miR-190b, hsa-miR-33a, hsa-miR-329, hsa-miR-221, hsa-miR-612, hsa-miR-3185, hsa-miR-3156-5p, hsa-miR-107, hsa-miR-664, hsa-miR-3657; (inhibitor of Ythdf2) hsa-mir-615-3p (MIRT040054), hsa-mir-106b-5p (MIRT044257), hsa-mir-1 (MIRT023842), miR-145, hsa-miR-3607-3p, hsa-miR-200a, hsa-miR-301a, hsa-miR-519a, hsa-miR-141, hsa-miR-130b, hsa-miR-181b, hsa-miR-301b, hsa-miR-3117-3p, hsa-miR-1236, hsa-miR-181a, hsa-miR-519c-3p, hsa-miR-551b, hsa-miR-519e, hsa-miR-519b-3p, hsa-miR-19b, hsa-miR-1303, hsa-miR-608, hsa-miR-145, hsa-miR-130a, hsa-miR-181c, hsa-miR-323b-3p, hsa-miR-421, hsa-miR-515-5p, hsa-miR-3666, hsa-miR-181d, hsa-miR-146a, hsa-miR-4295, hsa-miR-454, hsa-miR-3919, hsa-miR-19a, hsa-miR-543, hsa-miR-4262; (inhibitor of Ythdf3) hsa-miR-582-3p, hsa-miR-579, hsa-miR-520e, hsa-miR-520f, hsa-miR-3152-3p, hsa-miR-106a, hsa-miR-30d, hsa-miR-30a, hsa-miR-93, hsa-miR-508-5p, hsa-miR-29a, hsa-miR-3148,hsa-miR-490-5p,hsa-miR-520b,hsa-miR-20a,hsa-miR-409-3p,hsa-miR-4255,hsa-let-7i,hsa-miR-373,hsa-let-7e,hsa-miR-520c-3p,hsa-miR-3920,hsa-miR-127-5p,hsa-miR-380,hsa-miR-616,hsa-miR-4277,hsa-miR-448,hsa-miR-16-2,hsa-let-7c,hsa-miR-340,hsa-miR-373,hsa-miR-520a-3p,hsa-miR-144,hsa-miR-1265,hsa-miR-548x,hsa-miR-362-5p,hsa-miR-33b,hsa-miR-26b,hsa-miR-17,hsa-miR-569,hsa-miR-3618,hsa-miR-576-5p,hsa-miR-922,hsa-miR-302a,hsa-miR-106b,hsa-miR-888,hsa-miR-484,hsa-let-7b,hsa-miR-582-5p,hsa-let-7f,hsa-miR-30b,hsa-miR-524-5p,hsa-miR-302d,hsa-let-7d,hsa-miR-513a-5p,hsa-miR-500a,hsa-miR-570,hsa-miR-548l,hsa-miR-105,hsa-miR-374c,hsa-let-7g hsa-miR-372,hsa-miR-3658,hsa-let-7a,hsa-miR-3908,hsa-miR-302b,hsa-miR-526b,hsa-miR-190,hsa-miR-181b,hsa-miR-433,hsa-miR-98,hsa-miR-3606,hsa-miR-595,hsa-miR-548am,hsa-miR-187,hsa-miR-561,hsa-miR-181a,hsa-miR-3155,hsa-miR-655,hsa-miR-302c,hsa-miR-195,hsa-miR-26a,hsa-miR-590-3p,hsa-miR-30c,hsa-miR-502-5p,hsa-miR-495,hsa-miR-137,hsa-miR-181c,hsa-miR-520d-5p,hsa-miR-3942-5p,hsa-miR-202, hsa-miR-302e, hsa-miR-513c, hsa-miR-885-5p, hsa-miR-520a-5p, hsa-miR-583, hsa-miR-1297, hsa-miR-7-1, hsa-miR-520d-3p, hsa-miR-3155b, hsa-miR-3182, hsa-miR-519d, hsa-miR-550a, hsa-miR-7-2, hsa-miR-181d, hsa-miR-190b, hsa-miR-1912, hsa-miR-151-3p, hsa-miR-33a, hsa-miR-525-5p, hsa-miR-20b, hsa-miR-514b-5p, hsa-miR-30e, hsa-miR-4262, hsa-miR-636; (inhibitor of elF3) hsa-mir-92b-3p (MIRT040734), hsa-mir-16-5p (MIRT031705), hsa-mir-18a-3p (MIRT040974), hsa-mir-155-5p (MIRT020771), hsa-mir-484 (MIRT042324), hsa-let-7c-5p (MIRT051776), hsa-miR-3910, hsa-miR-148b, hsa-miR-136, hsa-miR-15a, hsa-miR-488, hsa-miR-500a, hsa-miR-1297, hsa-miR-3159, hsa-miR-374c, hsa-miR-424, hsa-miR-7-1, hsa-miR-186, hsa-miR-195, hsa-miR-15b, hsa-miR-26b, hsa-miR-505, hsa-miR-1206, hsa-miR-653, hsa-miR-1283, hsa-miR-7-2, hsa-miR-196a, hsa-miR-497, hsa-miR-33a, hsa-miR-655, hsa-miR-26a hsa-miR-16, hsa-mir-151a-3p (MIRT043600), hsa-mir-92a-3p (MIRT049064), hsa-mir-615-3p (MIRT039779), hsa-mir-877-3p (MIRT036964), hsa-mir-222-3p (MIRT046746), hsa-mir-423-3p (MIRT042468),hsa-mir-324-3p (MIRT042887), hsa-mir-124-3p (MIRT022932), hsa-miR-3140-3p, hsa-miR-124, hsa-miR-198, hsa-miR-525-5p, hsa-miR-506, hsa-miR-520a-5p, hsa-miR-196a*hsa-miR-3117-3p, hsa-mir-342-5p (MIRT038210), hsa-mir-378a-5p (MIRT043981), hsa-mir-615-3p (MIRT040086), hsa-let-7b-5p (MIRT052211), hsa-mir-455-3p (MIRT037879), hsa-miR-4267, hsa-miR-590-3p, hsa-mir-106b-5p (MIRT0443hsa-mir-103a-3p(MIRT027037),hsa-mir-1(MIRT023492),hsa-mir-19b-3p(MIRT031105),hsa-mir-100-5p(MIRT048400),hsa-mir-93-5p(MIRT02 7989),hsa-mir-16-5p(MIRT031379),hsa-let-7b-5p(MIRT052150),hsa-miR-520f,hsa-miR-300,hsa-miR-15a,hsa-miR-200a,hsa-miR-605,hsa-m iR-30d, hsa-miR-30a, hsa-miR-3613-3p, hsa-miR-509-3-5p, hsa-miR-34c-5p, hsa-miR-324-3p, hsa-miR-1248, hsa-miR-152, hsa-miR-548t, hsa-miR-4310, hsa-miR-145, hsa-miR-516a-3p, hsa-miR-16, hsa-miR-3668, hsa-miR-4277, hsa-miR-448, hsa-miR-16-2, hsa-miR-148b, hsa-miR-509-5p ,hsa-miR-103a,hsa-miR-1265,hsa-miR-2115,hsa-miR-548c-3p,hsa-miR-148a,hsa-miR-548p,hsa-miR-513a-3p,hsa-miR-497,hsa-miR-3647-3p,hsa-miR-382,hsa-miR-30b,hsa-miR-543,hsa-let-7f-2,hsa-miR-1269,hsa-miR-3164,hsa-miR-503,hsa-miR-500a,hsa-miR-449a,hsa-miR-141 ,hsa-miR-424,hsa-miR-3908,hsa-miR-889,hsa-miR-2116,hsa-miR-330-3p,hsa-miR-15b,hsa-miR-181b,hsa-miR-187,hsa-miR-1237,hsa-miR-449b,hsa-miR-101,hsa-miR-381,hsa-miR-618,hsa-miR-222,hsa-miR-181a,hsa-miR-432,hsa-miR-96,hsa-miR-19b,hsa-miR-195,hsa-miR-548n,hsa-miR-485-5p,hsa-miR-217,hsa-miR-30c,hsa-miR-495,hsa-miR-137,hsa-miR-1288,hsa-mi R-181c,hsa-miR-3942-5p,hsa-miR-548v,hsa-miR-487a,hsa-miR-221,hsa-miR-891b,hsa-miR- 205,hsa-miR-195,hsa-miR-4271,hsa-miR-3611,hsa-miR-516b,hsa-miR-181d,hsa-miR-154,hs a-miR-646, hsa-miR-153, hsa-miR-34a, hsa-miR-19a, hsa-miR-107, hsa-miR-30e and hsa-miR-4262. ,

[0207] According to one embodiment, the present invention includes an isolated mesenchymal stem cell population prepared by any of the methods described herein. For example, in certain embodiments, the present invention may include an expanded isolated mesenchymal stem cell population prepared by any of the expansion and / or separation methods described herein.

[0208] In one embodiment, a kit for isolating a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof is provided. The kit includes a system for contacting a biological sample containing MSCs with one or more N-cadherin antibodies and instructions for use thereof. In one embodiment, the kit further includes a system for isolating a population of mesenchymal stem cells (MSCs) for subsequent transplantation into a subject in need thereof. 6 A system for introducing a mutation into an MSC population to expand the MSC population, and instructions for use thereof. In another embodiment, the system for introducing a mutation into an MSC population comprises one or more reagents capable of introducing a mutation that results in deletion, replacement, or reduced expression of a gene expressing Ythdf2 into the MSC population. In yet another embodiment, the system for introducing a mutation into an MSC population comprises an Mx1-Cre targeting system that targets an mRNA-modified reader. 6 A mRNA modification reader is inactivated or deleted at least a portion of the gene. In one embodiment, the system for introducing mutations into the MSC population includes a method for delivering shRNA incorporated into the MSC population to reduce the expression of mRNA. 6 A lentiviral reagent for mRNA modification of gene expression.

[0209] According to another embodiment of the present invention, a method for administering mesenchymal stem cells (MSCs) to a subject in need thereof is provided. The method comprises isolating MSCs from a biological sample comprising an MSC population by contacting the biological sample with one or more N-cadherin antibodies, and administering the isolated MSCs to the subject. In addition, in one embodiment, the method further comprises administering to the subject a MSC that results in expression of m 6 A mutation that results in deletion, replacement, or reduced expression of a gene encoding a modified mRNA reader is introduced into a biological sample containing a population of MSCs, and the biological sample is cultured under suitable conditions and for a sufficient period of time to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject. In one embodiment, the mutation results in deletion of a gene expressing Ythdf2. In another embodiment, a shRNA that results in reduced expression of a gene expressing Ythdf2 is incorporated into the population of HSCs. According to another aspect, the MSCs are obtained from a biological sample comprising a tissue selected from umbilical cord blood, peripheral blood, and bone marrow.

[0210] According to another embodiment, a method for reconstructing bone marrow in a subject in need thereof is provided. The method comprises isolating MSCs from a biological sample containing a population of mesenchymal stem cells (MSCs) by contacting the biological sample with one or more N-cadherin antibodies, and administering the isolated MSCs to the subject. In addition, according to one aspect, the method further comprises administering to the subject a method that results in expression of m 6 A deletion, substitution, or reduced expression mutation of a gene of an mRNA modification reader is introduced into a biological sample comprising a population of MSCs, and the biological sample is cultured under suitable conditions and for a sufficient period of time to expand the number of MSCs in the sample to a number sufficient for transplantation into a subject. In one embodiment, the MSCs are obtained from a biological sample comprising a tissue selected from the group consisting of umbilical cord blood, peripheral blood, and bone marrow.

[0211] According to another embodiment, a method for treating a subject in need of a transplant selected from a bone marrow transplant, a peripheral blood transplant, and an umbilical cord blood transplant, comprising administering to the subject an isolated MSC population obtained by any of the methods described herein. According to an embodiment, the sample is from an autologous or allogeneic source. According to yet another embodiment, the sample is from an autologous source.

[0212] In the present invention, a "therapeutically effective amount" is an amount sufficient to produce a beneficial or desired result. With respect to the treatment of a mammal, a "therapeutically effective amount" of a modulator and / or expanded cells is an amount sufficient to treat, control, alleviate, ameliorate, or stabilize a condition, such as a bone marrow disorder, in the mammal. A therapeutically effective amount can be administered in one or more doses.

[0213] The therapeutically effective amount is generally determined by a physician on a case-by-case basis and is within the capabilities of those skilled in the art. Several factors are generally considered when determining an appropriate dosage. These factors include the age, sex, and weight of the subject, the disease being treated, the severity of the disease, and the form of the drug being administered.

[0214] Effective dosage form, mode of administration and dosage can be determined empirically, and it is within the skill of the art to determine this. It will be appreciated by those skilled in the art that dosage will vary with route of administration, excretion rate, duration of treatment, the nature of any other drug administered, age, size and species of the animal, and factors well-known to the medical and veterinary fields. Typically, the appropriate dose of a modulator according to the present invention will be the amount of the modulator, which is the minimum dose that effectively produces the desired effect. The effective dose of a modulator can be administered as two, three, four, five, six or more subdoses, which are administered separately at appropriate intervals throughout the day.

[0215] The modulators can be administered in any desired and effective manner: as a pharmaceutical composition for oral ingestion, or in any appropriate manner for parenteral or other administration, such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. In addition, the modulators of the present invention can be administered in conjunction with other treatments. If desired, the modulators of the present invention can be encapsulated or otherwise protected from gastric or other secretions.

[0216] Although the modulators of the present invention can be administered alone, they are preferably administered in the form of a pharmaceutical formulation (composition). Such pharmaceutical formulations typically contain one or more modulators mixed with one or more pharmaceutically acceptable carriers and optionally one or more other compounds, drugs, ingredients and / or materials as active ingredients. Regardless of the route of administration selected, the modulators of the present invention can be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).

[0217] Pharmaceutically acceptable carriers are well known in the art (see, for example, Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.) and The National Formulary (American Pharmaceutical Association, Washington, DC), and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate, and dibasic calcium phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethanol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn oil, germ oil, olive oil, castor oil, sesame oil, cottonseed oil, and peanut oil), cocoa butter, waxes (e.g., suppository wax), paraffin, silicones, talc, salicylates, and the like. Each pharmaceutically acceptable carrier used in a pharmaceutical composition comprising a modulator of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Suitable carriers for a chosen dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using one of ordinary skill in the art.

[0218] The pharmaceutical compositions comprising the modulators of the present invention may optionally contain other ingredients and / or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, hydroxypropylmethylcellulose, sucrose, and acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethylcellulose, and sodium carbonate; (5) solubilizers, such as paraffin wax; (6) absorption enhancers, such as quaternary ammonium compounds; (7) Wetting agents such as cetyl alcohol and glyceryl monostearate; (8) Absorbents such as kaolin and bentonite; (9) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) Suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth; (11) Buffers; (12) Excipients such as lactose, milk sugar, polyethylene glycol, animal and vegetable fats, oils, waxes, paraffin, cocoa butter, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, Bentonite, silicic acid, talc, salicylates, zinc oxide, aluminum hydroxide, calcium silicate and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surfactants; (16) dispersants; (17) release-controlling or absorption-delaying agents, such as hydroxypropyl methylcellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin and wax; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and suspending agents; (22) solubilizing agents and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, ethyl ester, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents that render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; (28) sweeteners, flavorings, colorings, aromas and preservatives. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a chosen dosage form and intended route of administration are well known in the art, and one of ordinary skill in the art can determine acceptable ingredients and materials for a chosen dosage form and method of administration.

[0219] Pharmaceutical compositions suitable for oral administration can be in the form of capsules, cachets, pills, tablets, powders, granules, solutions or suspensions in aqueous or non-aqueous liquids, oil-in-water or water-in-oil liquid emulsions, elixirs or syrups, lozenges, boluses, electuaries or pastes. These formulations can be prepared by methods known in the art, for example, by conventional pan coating, mixing, granulation or lyophilization methods.

[0220] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules, etc.) can be prepared by mixing the active ingredient with one or more pharmaceutically acceptable carriers and optionally one or more fillers, extenders, adhesives, wetting agents, disintegrants, dissolution retardants, absorption promoters, wetting agents, absorbents, lubricants and / or colorants. Suitable excipients can be used as fillers in soft and hard-filled gelatin capsules for similar solid compositions. Tablets can be made optionally with one or more auxiliary ingredients by compression or molding. Suitable adhesives, lubricants, inert diluents, preservatives, disintegrants, surfactants or dispersants can be used to prepare compressed tablets. Molded tablets can be prepared by molding in a suitable machine. Tablets and other solid dosage forms (e.g., dragees, capsules, pills and granules) can optionally be scored or prepared with coatings and shells (e.g., enteric coatings and other coatings well-known in the field of pharmaceutical formulations). They can also be formulated to provide a slow or controlled release of the active ingredient therein. They can be sterilized, for example, by filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of such a composition that they release the active ingredient only or preferentially in a specific part of the gastrointestinal tract, optionally in a delayed manner. The active ingredient may also be in microencapsulated form.

[0221] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. Liquid dosage forms may contain suitable inert diluents commonly used in the art. In addition to inert diluents, oral compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweeteners, flavorings, coloring agents, flavoring agents, and preservatives. Suspensions may contain suspending agents.

[0222] Pharmaceutical compositions for rectal or vaginal administration may be in the form of suppositories, which can be prepared by mixing one or more active ingredients with one or more non-irritating carriers that are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Pharmaceutical compositions suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically acceptable carriers as are known in the art.

[0223] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops, and inhalants. The active compound can be mixed with a suitable pharmaceutically acceptable carrier under aseptic conditions. Ointments, pastes, creams, and gels can contain excipients. Powders and sprays can contain excipients and propellants.

[0224] The pharmaceutical composition suitable for parenteral administration comprises one or more regulators combined with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions or sterile powders (which can be reconstituted as sterile injections or dispersions immediately before use), which may comprise suitable antioxidants, buffers, solutes (which make the preparation isotonic with the blood of the intended recipient) or suspending agents or thickening agents. Suitable fluidity can be maintained, for example, by using coating materials, in the case of dispersions, by maintaining the required particle size and by using surfactants. These compositions may also comprise suitable adjuvants, such as wetting agents, emulsifiers and dispersants. It may also be necessary to include isotonic agents. In addition, the extended absorption of injectable drug forms can be caused by comprising an agent that delays absorption.

[0225] In some cases, in order to prolong the effect of a pharmaceutical containing a modulator of the present invention, it is desirable to slow its absorption from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of the poorly water-soluble crystalline or amorphous material.

[0226] The absorption rate of the drug then depends on its dissolution rate, which in turn can depend on crystal size and crystalline form. Alternatively, delayed absorption of parenteral-administered drugs can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms can be prepared by forming a microcapsule matrix of the active ingredient in a biodegradable polymer. Depending on the ratio of the active ingredient to the polymer and the properties of the specific polymer used, the release rate of the active ingredient can be controlled. Injectable depot preparations can also be prepared by entrapping the drug in liposomes or microemulsions compatible with body tissues. The injectable material can be sterilized, for example, by filtering through a bacteria-retaining filter.

[0227] The formulations can be presented in unit-dose or multi-dose sealed containers such as ampoules and vials and can be stored in a lyophilized condition, requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the type described above.

[0228] The following examples are provided to further illustrate the methods and compositions of the present invention. These examples are illustrative only and are not intended to limit the scope of the present invention in any way. Example

[0229] The examples herein examine Ythdf2, a mRNA that is recognized to promote targeted mRNA decay. 6 A study was conducted (Wang et al. Blood, 505:17-120, 2014), at least in part to investigate its role in HSC maintenance. Without being limited to any particular theory, it is believed that manipulation of Ythdf2 could potentially affect a large number of m 6 Ythdf2 depletion specifically expands mouse and human HSCs without reversing lineage fate. Therefore, it is believed that Ythdf2 may play an important role in regulating HSC self-renewal and provides a new approach to enhance the ex vivo expansion of hUCB HSCs, including for clinical applications.

[0230] The Examples also demonstrate the functional definition of drug-resistant rHSC populations and the discovery that rHSCs primarily express N-cadherin during homeostasis and under chemotherapeutic stress. + The cells are maintained in the endosteal niche. N-cad is also shown in the endosteal region. + The cells are mesenchymal stem cells and contribute to the maintenance of rHSCs.

[0231] Example A

[0232] In the "A" examples below the following protocol was used.

[0233] Mice. Ythdf2 conditional KO mice were generated by the experimental groups of Chuan He and Bin Shen. Mice were housed in the animal facility of the Stowers Institute for Medical Research (SIMR) and handled according to institute and NIH guidelines. All procedures were approved by SIMR's IACUC.

[0234] Flow cytometry and HSPC sorting. Mouse HSPC, progenitor cells and lineage cells were harvested from BM (femur and tibia) and spleen. Red blood cells were lysed using 0.16M ammonium chloride solution, and cells were filtered with a 70 μm filter to produce a single cell suspension. In order to identify mouse HSC, cells were stained with antibodies against Sca-1 (D7), c-Kit (2B8), CD34 (RAM34), Flk2 (A2F10), CD48 (HM48-1), CD150 (TC15-12F12.2) and using a lineage mixture including CD3 (145-2C11), CD4 (RM4-5), CD8 (53-6.7), Mac-1 (M1 / 70), Gr1 (RB6-8C5), CD45R (B220, RA3-6B2), IgM (Il-41) and Ter119 (TER-119). For progenitor and lineage cells, cells were stained with antibodies as previously described (Qian, P. et al. Cell Stem Cell, 18:214-228, 2016, doi:10.1016 / j.stem.2015.11.001). Dead cells were excluded using 7-aminoactinomycin D (7-AAD) (A1310, Life technologies). Human umbilical cord blood samples were collected from St. Louis Cord Blood Bank. Lymphoprep TM (StemCell technologies) to separate mononuclear cells, and then human CD34 was isolated using the Human CD34 MicroBead Kit UltraPure (Miltenyi Biotec). + Umbilical cord blood cells. To quantify human HSPCs, cells were stained with antibodies against CD34 (581), CD38 (HIT2), CD45RA (HI100), CD90 (5E10), CD49f (GoH3), and EPCR / CD201 (RCR-401). Cell sorting and analysis were performed on a MoFlo (Dako), InFlux cell sorter (BD Biosciences), and / or MACSQuant (Miltenyi Biotec). Data were analyzed using FlowJo software.

[0235] Homing assay. In vivo homing assay was performed as previously described (He et al. Methods in Molecular Biology, 1185: 279-284, 2014). Basically, whole bone marrow (WBM) cells from CD45.2 mice were labeled with 5 μM 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFDA SE) (Molecular Probes) at 37°C for 10 minutes, washed three times, and then 1×10 6 WBM was transplanted into lethally irradiated PTPRC mice. 18 hours later, femurs and tibias were flushed and CFDA SE+ cells were determined.

[0236] Competitive reconstitution assay. 2 × 10 5 , 7.5×10 4 or 2.5×10 4 Donor-derived WBM cells and 2×10 5 Competitive reconstitution assays were performed by intravenously transplanting 10 rescued cells (CD45.1) into ten lethally irradiated (10 Gy) PTPRC recipient mice. For secondary transplantation, primary transplant recipients were sacrificed. BM cells were isolated from femurs and tibias and then inoculated at 1 × 10 6 The cells were transplanted into irradiated secondary recipient mice at a dose of 100 mg / dL. Baytril water was administered to the recipient mice three days before irradiation and continued for two weeks after irradiation. Primary and secondary CRU frequencies were measured using ELDA software (Hu et al. Journal of Immunological Methods, 347:70-78, 2009), where successful engraftment was defined as the presence of unique CD45.2 cells in the peripheral blood, accounting for ≥5% and ≥1% of total hematopoietic cells, respectively. + CD45.1 - In addition, secondary transplant recipient mice that died before 16 weeks after transplantation were counted as failed engraftments.

[0237] Cell cycle and apoptosis assays. Cell cycle analysis was performed using a FITC mouse anti-human Ki67 kit (BD Pharmingen) according to the manufacturer's instructions. Briefly, 5 × 10 6BM cells were stained with HSC antibodies. Cells were fixed with 4% paraformaldehyde at 4°C overnight or at room temperature (RT) for 1 hour and then permeabilized with 0.2% Triton X-100 for 15 minutes on ice. Cells were washed with PBS containing 2% FBS and then incubated with Ki-67 antibody for 1 hour at room temperature in the dark, followed by incubation with SYTOX Red (Invitrogen) for another 5 minutes at room temperature and then subjected to flow cytometric analysis using an InFlux cell sorter (BD Biosciences). For apoptosis analysis, 5 × 10 6 BM cells were stained with Annexin V (Invitrogen) and SYTOX Red.

[0238] m 6 A RNA-IP-seq. 10 cells from C57BL / 6J mice were 5 LT-HSC, ST-HSC (LSK CD34 + FLK2 - ) and MPP (LSK CD34 + FLK2 + Two replicates of ) were sorted into TRIzol (Invitrogen) and total RNA was isolated according to the manufacturer's instructions. RNA was fragmented into fragments of approximately 100 nucleotides using Ambion fragmentation reagent (incubation at 70°C for 2 minutes). The samples were then Turbo DNase treated (Ambion) followed by phenol-chloroform extraction and resuspended in 85 μl of nuclease-free water, and 5 μl was saved as input. The remaining 80 μl of RNA fragments were then diluted into IPP buffer (150 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5). The RNA was mixed with 25 μl of pre-bound 3 μg of anti-m 6Protein G magnetic beads with monoclonal antibody A (Synaptic Systems) were incubated in IPP buffer at 4°C for 3 hours. The beads were washed twice with 200 μl of IPP buffer, twice with 200 μl of low salt buffer (50 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5), and twice with 200 μl of high salt buffer (500 mM NaCl, 0.1% NP-40, 10 mM Tris-HCl, pH 7.5). The beads were then treated with 300 μl of elution buffer (5 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.05% SDS, 4.2 μl of proteinase K (20 mg / ml)) at 50°C for 1.5 hours, and RNA was recovered by phenol:chloroform extraction followed by ethanol precipitation. Triplicate human CD34 was isolated as described above. + Umbilical cord blood cells were isolated and total RNA was isolated using TRIzol. RNA was fragmented into fragments of approximately 100 nucleotides using Ambion fragmentation reagent (incubation at 70°C for 2 minutes and 50 seconds). The samples were then treated with Turbo DNase (Ambion) followed by phenol:chloroform extraction and resuspended in 18 μl of nuclease-free water, and 1 μl was saved as input. Following the manufacturer's instructions, the RNA was fragmented using Methyladenosine enrichment kit 6 A RNA IP.

[0239] In RNA m 6 After A preparation, quality was assessed on an Agilent 2100 Bioanalyzer, and RNAseq libraries were generated using 1 ng (mouse) or 10 ng (human) RNA using 16 cycles (mouse) or 13 cycles (human) PCR2 amplification according to the manufacturer's instructions for the SMARTer Stranded Total RNA-Seq Kit–Pico Input Mammalian (Takara Bio Inc). This method uses random primers and template-switching oligonucleotides to generate complementary DNA, followed by ligation of barcoded adapters; ribosomal-derived cDNA is then removed by probe-directed enzymatic cleavage and subsequent enrichment of uncleaved fragments.

[0240] The protocol was modified to retain lower molecular weight sample fragments by using 1.2x SPRI bead concentration for PCR1 cleanup. To remove dimerized adapters, the library was size-selected between 160 and 600 bp using a Pippin Prep (Sage Science) 2% gel. The quality and quantity of the resulting library were checked using a bioanalyzer and a Qubit fluorometer (Life Technologies). Equimolar libraries were then pooled and re-quantified. For mouse m 6 A-seq, using HiSeq control software 2.2.58 on an Illumina HiSeq 2500 instrument, was used to sequence the libraries into 50 bp single sequence fragments. After sequencing, Illumina Primary Analysis version RTA 1.18.64 and Secondary Analysis version bcl2fastq2v2.18 were run to demultiplex the sequence fragments of all libraries and generate FASTQ files. 6 For A-seq, libraries were sequenced as single 75 bp reads on an Illumina NextSeq instrument using NextSeq Control Software 2.1.2. Following sequencing, Illumina Primary Analysis version NextSeq RTA 2.4.11 and Secondary Analysis version bcl2fastq2v2.18 were run to demultiplex the sequenced fragments from all libraries and generate FASTQ files.

[0241] Plasmid construction and stable cell line generation. Mouse Ythdf2 (mYthdf2) was cloned from a commercial cDNA clone (ORIGENE#MC200730) into the pcDNA5 / FRT / Flag plasmid using the following primers: mYthdf2 ORF clone BamhI F: 5'-CGC GGA TCC TCG GCC AGC AGC CTC TTG GA-3' and mYthdf2 ORF clone NotI R: 5'-ATA AGAATG CGG CCG CCT ATT TCC CAC GAC CTT GAC GT-3'. Flag-mYthdf2 was then subcloned into the EF1a promoter in the pSicoR-EF1a-IRES-EGFP lentiviral construct (Gibson et al., 2012). Forward primer: 5'-GTC GAC GGT ACC GCG GGC CCA TGG ATT ACA AGG ATG ACG ACG-3' and reverse primer: 5'-GAG GGA GAG GGG CGG ATC CCC TAT TTC CCA CGA CCT TGA CGT-3'. Human Ythdf2 (hYthdf2) was cloned from a plasmid provided by the laboratory of Chuan He using the following primers: forward 5'-CGT TCG AAA TGT CGGCCA GCA GCC TCT-3'; reverse 5'-TCC CCC GGG TTA TTT CCC ACG ACC TT-3'. hYthdf2 was then cloned into the pSicoR-EF1a-IRES-EGFP construct under the EF1a promoter by BstBI and XmaI restriction enzyme digestion and ligation. To generate the Flag-mYthdf2 HPC7 stable cell line, lentivirus was generated by transfecting the pSicoR-EF1a-Flag-mYthdf2-IRES-EGFP construct into 293T cells together with psPAX2 and pMD2.G plasmids at a ratio of 10:7.5:2.5 using calcium phosphate transfection. Viral particles were harvested 48, 72, and 96 hours after transfection, filtered with a 0.45 micron filter (Millipore), and then centrifuged at 18,000 RPM and 4°C for 2 hours. HPC7 cells were infected with the recombinant lentiviral transduction units in the presence of 4 μg / mL polyethylene (Sigma). 48 hours after infection, GFP was expressed. + Cells were sorted and cultured for experiments.

[0242] irCLIP-seq and data analysis. For irCLIP-seq, the procedure from previously reported methods (Zarneger et al. Nature Methods, 13:489-492, 2016); Simsek et al. Cell, 169:1051-1065e1018, 2017) was modified. Briefly, the irCLIP-seq was performed at 0.4 J / cm 2 UV crosslinked cells 3 times for about 3×10 8irCLIP was performed on HPC7 cells expressing Flag-Ythdf2. Whole-cell lysates were generated in lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 400 U / ml RNase inhibitor; 1 ml cell pellet and 2 ml lysis buffer). After pipetting up and down several times, the mRNP lysate was incubated on ice for 5 minutes and then snap-frozen with liquid nitrogen at -80°C. The mRNP lysate was thawed on ice and centrifuged at 15,000 g for 15 minutes to clear the lysate. Flag-Ythdf2 was isolated using 30 μl of protein G magnetic beads pre-conjugated with 2 μg of anti-Flag monoclonal antibody (Sigma) per 1 ml of lysate, rotating at 4°C for 2 hours. The beads were collected and washed eight times with 1 ml of ice-cold NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 200 U / ml RNase inhibitor) and once with 200 μl of irCLIP NT2 buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM MgCl2; 0.0005% NP-40). The mRNP complex was digested with 0.4 U / μl RNase 1 (Thermo Fisher #AM2294) in irCLIP NT2 buffer (30 μl of water supplemented with 6 μl of PEG400 (final concentration 16.7%)). The nuclease reaction was incubated in an Eppendorf Thermomixer at 30°C for 15 minutes, 1400 rpm for 15 seconds, and allowed to stand for 90 seconds. Nuclease digestion was terminated by adding 0.5 mL of ice-cold high stringency buffer (20 mM Tris, pH 7.5; 120 mM NaCl; 25 mM KCl; 5 mM EDTA; 1% Trition-X100; 1% sodium deoxycholate). The immunoprecipitate was then quickly rinsed with 0.25 mL followed by 0.05 mL of ice-cold irCLIP NT2 buffer. Ligation of irCLIP adapters and library construction followed a previously reported protocol (Zarneger et al. Nature Methods, 13: 489-492, 2016).

[0243] The data were demultiplexed using FAST-iCLIP version 0.9.3 and aligned to the mouse genome mm10 from UCSC using STAR (2.4.2a) with the parameters "--outFilterScoreMinOverLread 0--outFilterMatchNminOverLread 0--outFilterMatchNmin 0". RPM-normalized genome browsing tracks were created in R (3.4.1) and plotted using the Gviz software package (1.20.0). Enriched motifs were identified by taking the midpoint of each binding site found in all three replicates, adding 20 bases upstream and downstream, and running MEME (4.11.1) with the parameters "-dna-mod zoops-revcomp-minw 5-maxw 10-nmotifs 10-maxsize 1000000". After identifying the motifs, we ran tomtom (4.11.1) against transfac (1-2017) to identify known binding sites. GO enrichment analysis was performed using the hypergeometric test in R. GO terms were considered enriched if the BH-adjusted p-value was less than 0.05. Selected terms of interest are shown in a bar chart. The bars in the bar chart represent the percentage of genes with that term in the tested list divided by the percentage of genes with that term in the genome. Peaks found by FAST-iCLIP in all three replicates were assigned to various features in the genome. Promoters were defined as 150 bases upstream of the TSS. "Trans_stop" was defined as 200 bases upstream and downstream of the transcript start site.

[0244] Umbilical cord blood transduction. Umbilical cord blood transduction was performed as previously described (Rentas, S. et al. Nature, 532:508-511, 2016, doi:10.1038 / nature17665). Briefly, fresh CD34 T cells were pre-stimulated in StemSpan medium supplemented with the growth factors interleukin 6 (IL-6; 20 ng / ml, Peprotech), stem cell factor (SCF; 100 ng / ml, Peprotech), Flt3 ligand (FLT3-L; 100 ng / ml, Peprotech), and thrombopoietin (TPO; 20 ng / ml, Peprotech). + Umbilical cord blood cells or flow cytometry-sorted CD34 + CD38 -Cells were transduced for 12-18 hours. Lentivirus was then added to the same culture medium at a multiplicity of infection (MOI) of 50-200 for 24 hours. Cells were then maintained for 2 days after transduction before in vitro or in vivo assays. Human YTHDF2 was knocked down by targeting shRNA targeting 5'-AAGGACGTTCCCAATAGCCAA-3' near the N-terminus of the CDS, as used in a previous report (Wang, X. et al. Nature 505:117-120, 2014, doi:10.1038 / nature12730). A scrambled shRNA (seed sequence 5'-GCGCGATAGCGCTAATAAT-3') was used as a control.

[0245] Clonogenic progenitor cell assay. GFP was flow-sorted from transduced cells (12,000 cells / ml) on day 10 of culture. + Umbilical cord blood cells were resuspended in semisolid methylcellulose medium (Methocult H4034; StemCell Technologies) and colonies were counted after 14 days of culture.

[0246] Human umbilical cord blood HSPC culture. 2 days after transduction, human umbilical cord blood CD34 + or CD34 + CD38 - cells and GFP was measured by flow cytometry + To ensure that the same number of GFP cells were cultured before amplification + cells, and GFP - Cells were added to the + Percentage of cells with matching GFP between control and hYthdf2 KD + Then the cells were cultured at 10 5 Cells were plated at a density of 100 μg / ml in StemSpan medium (StemCell Technologies) supplemented with growth factors IL-6 (20 ng / ml), SCF (100 ng / ml), FLT3-L (100 ng / ml), TPO (20 ng / ml) and CHIR99021 (250 nM) (Stemgent) (Perry et al. Genes and Development, 25: 1928-1942, 2011).

[0247] Human HSC xenografts. For ex vivo expansion analysis of human umbilical cord blood HSCs, cells were transduced with human YTHDF2 shRNA or control shRNA for 10 5 sorted CD34 + CD38 -Cells were cultured for 3 days and then the transduction efficiency (% GFP - / + ) and stem cell markers. On day 10, cultured cells were collected for stem cell marker analysis. For the initial LDA assay of hUBC HSCs, CD34 + Cells were transduced with human YTHDF2 shRNA or control shRNA at an MOI of 50. The medium was changed 24 hours after infection. Three days after infection, equal numbers of GFP were sorted from control or YTHDF2 KD cells. + The cells were cultured overnight. Three doses of GFP, 50K, 20K and 10K, were added. + The cells were transplanted into sublethally irradiated (3.25 Gy) NSG mice. The cutoff value for HSC engraftment was total CD45 in the BM of primary transplant recipients. + Cells showing more than 1% human CD45 + GFP + For hUCBHSC secondary LDA analysis, BM cells from the top two dosed primary recipients were collected and pooled together 10 weeks after transplantation. 1.2 × 10 7 , 8×10 6 , 4×10 6 Three doses of BM cells were transplanted into sublethally irradiated (3.25 Gy) NSG mice. The cutoff value for HSC engraftment was the total CD45 + Cells showing more than 0.2% human CD45 + GFP + HSC frequency was assessed using ELDA software (Hu et al., Journal of Immunological Methods, 347: 70-78, 2009). For all human cord blood xenotransplantation experiments, 6-8 week old female NSG mice were used.

[0248] m 6 A-seq data analysis. 6 A-seq data were aligned with the transcriptomes of hg19 and mm10. 6 Peak A, hg19 and mm10 transcriptomes were divided into 25 nucleotide wide tiles. 6 The number of sequenced fragments in AIP and non-IP (control) samples was counted, and p-values ​​were calculated using Fisher's exact test and adjusted for multiple testing. 6The tiles with A signal enrichment (adjusted -Pval <= 0.05) were merged into larger regions. Regions smaller than 100 bp were discarded, and regions larger than 200 bp were divided into sub-regions of 100 to 200 bp; m was calculated relative to the control in each region. 6 A signal; regions with at least 2-fold enrichment in all replicates were identified as m 6 Peak A. By all m 6 A peak overlapped with hg19 and mm10 RefGene annotations to determine m 6 A peak distribution and m 6 A marker gene. 6 A peak overlaps with hg19RefGene to identify m 6 A marker gene. To filter transcription factors, all three samples were analyzed by m 6 The genes marked by A were compared with the human transcription factor database http: / / fantom.gsc.riken.jp / 5 / sstar / Browse_Transcription_Factors_hg 19. GO term analysis was then performed using the R package GO enrichment. 6 A-tagged human transcription factors were used as the search list, and all expressed genes were used as background. BP terms with significant enrichment for hematopoietic-related BPs were used to generate Figure 3C .

[0249] RNA-seq. Human umbilical cord blood CD34 cells were transduced with control or human YTHDF2 KD lentivirus. + cells and GFP was sorted after 10 days + CD34 + For each group, 12,000 GFP + CD34 +Three replicates of cells were prepared and used to extract total RNA. Four nanograms of high-quality total RNA was used for cDNA synthesis and library preparation using SMART-Seq v4Ultra Low Input RNA Kit for Sequencing (Takara, 634891) and Nextera XT (Illumina, FC-131-1096) according to the manufacturer's instructions. The quality and quantity of the resulting short fragment library were checked using an Agilent 2100 bioanalyzer and an Invitrogen Qubit fluorometer. In the high-output flow cell of the Illumina NextSeq 500 instrument, equimolar libraries were merged, requantified, and sequenced into single sequencing fragments of 75 base pairs. After sequencing, Illumina Primary Analysis version NextSeq RTA 2.4.11 and Secondary Analysis version bcl2fastq2 2.18 were run to multiplex the sequencing fragments of all libraries and generate FASTQ files.

[0250] For RNA-seq analysis, the sequenced fragments were aligned to the UCSC genome hg38 using the Ensembl 87 gene model using Tophat version 2.0.13 with default parameters. Sequencing fragment counts were generated using HTSeq-count with -mintersection-nonempty. Sequencing fragments were also aligned to the ERCC control sequence and the counts were tabulated. Scaling factors were calculated based on the median ERCC counts for each sample and used for normalization. Differentially expressed genes were found using the edgeR package (3.18.1) in R (3.4.1). Differentially expressed genes were required to have a BH-adjusted p-value <.05 and a 2-fold expression change.

[0251] RNA stability assay. 15,000 sorted LT-HSCs, ST-HSCs, and MPPs were cultured in StemSpan SFEM medium (Stem Cell Technologies) (Perry et al. Genes and Development, 25: 1928-1942 (2011)) supplemented with 10 μg / mL heparin (Sigma), 0.5× penicillin / streptomycin (Sigma), 10 ng / mL recombinant mouse (rm) SCF (Biovision, Inc.), and 20 ng / mL Tpo (Cell Sciences, Inc.) at 37°C 5% CO2 5% O2. Sorted cells were treated with 5 μM actinomycin D (Sigma) to inhibit mRNA transcription. Cells were harvested 0 or 4 hours after treatment, and total RNA was extracted and used for RNA-seq.

[0252] m 6 A RNA methylation quantification.

[0253] Mouse BM lineage-negative cells from wt and Ythdf2 KO mice were enriched using a mouse lineage cell depletion kit (Miltenyi Biotec), and total RNA was extracted using TRIzol (Invitrogen). 6 ARNA methylation quantification kit (Abcam ab185912) was used to quantify Lin - m in cells 6 A. RNA methylation was quantified using 200 ng of total RNA per replicate per group.

[0254] qPCR analysis.

[0255] Ten cells were sorted from wt and Ythdf2 KO mice. 5 LSK cells. Total RNA was extracted using TRIzol (Invitrogen). cDNA was synthesized using a high-capacity RNA-to-cDNA TM Kit (Thermo) was used according to the manufacturer's protocol. The qPCR primers used are listed in Table S5. qPCR primers were used to verify the expression levels of transcription factors in wt and Ythdf2 KO HSPCs.

[0256] Western blotting and intracellular staining. To verify the KO or KD efficiency in the Ythdf2 KO mouse model or hUCB, 33,000 cKit were sorted from BM or transfected hUCB samples, respectively. + cells or 120,000 GFP + Cells. Figure 14BAs shown, overexpression efficiency was verified using HeLa cells transduced to overexpress human YTHDF2. Immunoblotting was performed using an anti-YTHDF2 rabbit polyclonal antibody (MBL, RN123PW) and a β-actin mouse monoclonal antibody (NOVUS, NB600-501). Secondary antibodies used were IRDye 800CW goat anti-mouse IgG and IRDye 800CW goat anti-rabbit IgG antibodies (LI-COR). For intracellular staining, BM cells from wt and Ythdf2 KO mice were stained with HSC markers as described above and then fixed using the Cytofix / Cytoperm kit (BD Biosciences) according to the manufacturer's instructions. Fixed and permeabilized cells were immunostained with anti-YTHDF2 antibody (MBL RN123PW), anti-TAL1 antibody (Santacruz sc-393287), anti-GATA2 antibody (Santacruz sc-267), anti-RUNX1 antibody (Santacruz sc-365644), anti-STAT5 antibody (Santacruz sc-74442) and detected with Alexa-488 donkey anti-rabbit IgG antibody (Invitrogen).

[0257] Single cell immunostaining. 10,000 LSKs from wt and Ythdf2 KO mice were sorted onto poly-L-lysine-coated slides, placed in a humidified chamber and incubated at 4°C for 30 minutes to allow the cells to settle onto the slides. The cells were fixed with cold methanol for 10 minutes at room temperature, blocked with universal blocking reagent (BioGenex) for 30 minutes at room temperature, and stained overnight at 4°C with mouse TAL1 antibody (Santa Cruz, SC393287) or mouse IgG control (Abcam). The cells were then stained with Alexa Fluor 488 donkey anti-mouse IgG (Thermo Fisher Scientific) for 30 minutes at 4°C. Images were taken on a PerkinElmer Ultraview spinning disk system with a Yokagawa CS-X1 disk. All emitted light was collected onto a C9100-23 Hamamatsu EM-CCD using Velocity software (PerkinElmer). For Z stacks, the step size was set to 400 nm. The staining intensity of each image was quantified by ImageJ program.

[0258] FISH combined with fluorescent immunostaining. Using Cytospin TMSorted LSK were spun onto microscope slides (Fisher Scientific catalog number 12-544-4) using a 4-well centrifuge (Thermo Scientific catalog number A78300003) at 800 rpm for 1 minute at medium acceleration and then immediately immersed in 4% PFA (diluted from a 16% (wt / vol) aqueous solution, Electron Microscopy Sciences, catalog number 15710). The cells were fixed for 30 minutes at room temperature (25±2°C). RNA in situ hybridization was performed using the RNAscope Multiplex Fluorescence Detection Kit according to the manufacturer's instructions (Advanced Cell Diagnostics), with some modifications: antigen retrieval was not required and digestion was performed with a 1:15 diluted proteinase III solution at room temperature for 10 minutes. RNAscope probes targeting mouse Tal1 and Gata2 were designed and produced by ACDbio. After in situ hybridization, the slides were rinsed twice with PBST and directly subjected to background blocking (Background buster solution, Innovex, catalog number NB306) and primary antibody incubation. Anti-YTHDF2 (MBL, 1:500) and anti-Dcp1α (Santa Cruz, SC100706, 1:200) antibodies were diluted in antibody dilution reagent buffer (Life Technologies, catalog number 003118) and incubated overnight at 4°C. Donkey anti-rabbit Alexa Fluor 488 (Invitrogen, 1:500) and donkey anti-mouse Alexa Fluor 633 (Invitrogen, 1:500) were used for protein target multiplexing.

[0259] Example A-1

[0260] Ythdf2 KO leads to increased phenotypic HSCs in naive mice.

[0261] To investigate the effect of Ythdf2 on HSC phenotype, Crispr-Cas9 technology was used to generate Ythdf2 f / f Conditional knockout mice were then crossed with Mx1-Cre mice to specifically reduce Ythdf2 expression in hematopoietic cells (hereafter Ythdf2KO mice) ( Figure 1A and B). In the absence of pI:pC, BM HSPCs showed no discernible differences ( Figure 7A Four weeks after pI:pC injection, long-term HSCs (Lin) in Ythdf2 KO mice were significantly increased compared with those in wild-type (wt) littermates. - Sca1 + cKit + (LSK)CD34- Flk2 - ; LT-HSC) and short-term HSC (LSK CD34 + Flk2 - A significant increase was observed in the frequency and absolute number of SCs (ST-HSCs), but not in the multipotent progenitors (LSK CD34 + Flk2 + ; MPP) was not observed ( Figures 1C to 1E ). It was found that the frequency and absolute cell number of long-term HSC (LT-HSC) and ST-HSC increased more than 2-fold, while MPP showed a more moderate response ( Figure 1C and 1E Although Ythdf2 KO resulted in an increase in BM cellularity, the absolute numbers of committed progenitors, including common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), and common lymphoid progenitors (CLPs), as well as mature lineage cells, erythroid cells, myeloid cells, B cells, and T cells, were not significantly different between Ythdf2 KO and wt mice ( Figure 1F To H). Cell cycle analysis showed no significant changes in the quiescence of HSCs or MPPs after Ythdf2 KO ( Figure 7B Notably, the percentage of apoptotic cells in Ythdf2 KO LT-, ST-HSCs, and MPPs was significantly reduced compared with wt controls ( Figure 7C To further identify any potential HSC defects in Ythdf2 KO mice, the numbers of HSCs, committed progenitors, and mature lineages in the spleen were examined, and no significant differences were found between wt and Ythdf2 KO mice ( Figures 7D to 7H In conclusion, Ythdf2 KO in naive mice specifically increases HSC numbers without bias or defects in either progenitor or lineage cells.

[0262] Example A-2

[0263] Ythdf2 KO expands functional HSCs in mice.

[0264] To determine whether Ythdf2 KO expands functional HSCs, we first cultured 1 × 10 6 Carboxyfluorescein diacetate succinimidyl ester (CFDA SE)-labeled BM cells were transplanted into lethally irradiated recipient mice for short-term homing assays, and no significant differences were found in homing ability between mutant and wild-type controls ( Figure 7I ). Then, by transplanting 2×10 5 , 7.5×10 4 or 2.5×104 Donor BM cells (CD45.2) and 2×10 from the ptprc mutant strain 5 Recipient BM cells (CD45.1) were injected into lethally irradiated recipient mice for limiting dilution competitive repopulation unit assay (LDA) ( Figure 2A Consistent with the increased number of phenotypic HSCs in Ythdf2 KO mice, we found that the competitive repopulating units (CRU) in Ythdf2 KO HSCs were increased by 2.2-fold compared with controls ( Figure 2B ). In 2×10 5 In the Ythdf2 KO group, the overall repopulation rate of Ythdf2 KO donor cells was significantly increased compared with the control group 16 weeks after transplantation ( Figure 2C Furthermore, recipients of Ythdf2 KO BM cells exhibited significantly higher frequencies and absolute numbers of donor-derived LT-HSCs and ST-HSCs, but not MPPs, in BM compared with controls ( Figure 2D and 2E Furthermore, no significant changes were found in the number of donor-derived committed progenitors and mature lineages in the BM of transplant recipients from mutant and wt cells ( Figure 2F and 2G To determine the long-term repopulation capacity of HSCs from Ythdf2 KO mice, secondary transplantation using BM cells derived from primary recipients was performed. Notably, it was found that CRU from Ythdf2 KO cells showed a 3.5-fold increase compared to controls ( Figure 2H ) and showed no signs of leukemia in both BM and spleen at 16 weeks after secondary transplantation ( Figures 9A to 9F ). In addition, Figure 2I and 2J In a limited dilution assay, researchers transplanted total bone marrow (BM) from WT or Ythdf2 KO mice at three different doses. Four weeks after transplantation, they observed an increase in donor cell engraftment in mice transplanted with 200,000 Ythdf2 KO BM cells compared to WT BM cells. Furthermore, this increase did not produce a lineage bias in the transplant recipients.

[0265] We also investigated the long-term effects of Ythdf2 KO on hematopoiesis under steady-state conditions by examining stem, progenitor, and lineage cells in the BM and spleen over 5 months after pI:pC injection ( Figure 9A Although we observed a modest increase in LT-HSCs in the BM from Ythdf2KO mice compared with controls ( Figure 9C ), but no significant differences were found between Ythdf2 KO and control mice in progenitor and lineage cells from BM or spleen ( Figures 9D to 9JThese observations suggest that long-term Ythdf2 KO in vivo neither reverses lineage differentiation nor promotes aberrant proliferation, which is consistent with previous reports that Ythdf2 is not required for leukemogenesis. To verify the frequency of functional HSCs in BM 5 months after pI:pC induction, we isolated 7.5 × 10 HSCs from wt and Ythdf2 KO mice. 4 BM cells were transplanted into lethally irradiated recipients along with competent cells. We found that Ythdf2 KO resulted in significantly higher engraftment in recipients compared with wt controls, indicating that Ythdf2 KO has a long-term capacity for mouse HSC expansion in vivo ( Figure 9K Taken together, these data demonstrate that Ythdf2 KO leads to specific and significant mouse HSC expansion in vivo without affecting lineage commitment.

[0266] Example A-3

[0267] Ythdf2 by m 6 A-mediated mRNA decay regulates the expression of HSC self-renewal genes.

[0268] To explore the potential mechanism of how Ythdf2 KO expands HSCs, methylated RNA immunoprecipitation combined with high-throughput sequencing (MeRIP-seq or m 6 A-seq) was used to characterize mRNA expression in LT-HSCs, ST-HSCs, and MPPs isolated from adult C57BL / 6J mice. 6 A methylome was mapped (Meyer et al. Cell, 149: 1635-1646, 2012; Schwartz et al. Cell, 155: 1409-1421, 2013; Dominissini et al. Nature, 485: 201-206, 2012). m was selected by identifying significantly enriched overlapping peaks from two independent replicates. 6 Peak A. Consistent with previous studies (Meyer et al. Cell, 149:1635-1646, 2012); Dominissini et al. Nature, 485:201-206, 2012), we found that in all three HSPC populations, mRNAs were enriched in the mRNA open reading frame (ORF), in the 3' untranslated region (UTR), and near the stop codon. 6 Peak A. Transcripts of moderately expressed genes are more likely to be methylated ( Figures 10A to 10C ). Interestingly, it has been found that m 6The A modification is enriched in the mRNAs of transcription factors such as Gata2, Etv6, Stat5, and Tal1, which have been shown to be essential for HSC self-renewal and maintenance of the stem cell state (Wang et al. Blood, 113:4856-5865, 2009; Ebina et al. The EMBO Journal, 34:694-709, 2015; Orkin et al. Cell, 132:631-644, 2008; de Pater et al. The Journal of Experimental Medicine, 210:2843-2850, 2013; Hock et al. Genes and Development, 18:2336-2341, 2004; Lim et al. The Journal of Clinical Investigation 122:3705-3717, 2012; Reynaud et al. Blood, 106:2318-2328, 2005; and Kato et al. The Journal of Experimental Medicine, 210:2843-2850, 2013). Medicine, 202:169-179, 2005), indicating that m 6 A modification may play a key role in the regulation of HSC (Table S1, in HSPCs through m 6 A marks key transcription factors that are essential for HSC self-renewal and maintenance). Considering the increasing evidence that m 6 A mRNA methylation regulates stem cell fate determination by promoting the decay of mRNAs encoding transcription factors and genes in key signaling pathways involved in self-renewal and differentiation (Batista et al. Cell Stem Cell, 15:707-719, 2014; (Geula et al. Science 347:1001-1006,2015;Yoon et al. Cell,2017;Zhang et al. Nature,549:273-276,2017;Zhao et al. Nature,542:475-478,2017;Li et al. Cancer Cell,31:127-141,2017;Li et al. Nature,548:338-342,2017), next, the degradation rate of mRNA was measured in LT-HSC, ST-HSC and MPP by monitoring mRNA levels after transcriptional inhibition with actinomycin D. It was found that in ST-HSC and MPP, the degradation rate of methylated mRNA was significantly faster than that of unmethylated mRNA ( Figure 10D Because Ythdf2 is a well-established mRNA mediator of mRNA decay 6A "reader" (Wang et al. Nature, 505: 117-120, 2014), further identified targets of Ythdf2 by infrared UV cross-linking immunoprecipitation sequencing (irCLIP-seq) in the mouse multipotent hematopoietic precursor cell line HPC-7 (Pinto do et al. The EMBO Journal, 17: 5744-5756, 1998; Zarnegar et al. Nature Methods, 13: 489-492, 2016) ( Figure 3A ; Figures 11A to 11C The results showed that 57.8% of Ythdf2 target mRNAs contained m 6 Peak A Figure 11D ). Ythdf2 binding sites are enriched in conserved m 6 A motif and exhibits m 6 A distribution characteristics ( Figure 3B and 3C Gene ontology (GO) analysis of Ythdf2 target transcripts showed enrichment of genes related to hematopoietic or lymphoid organ development, indicating that Ythdf2 is involved in the regulation of hematopoiesis ( Figure 3D ). It is worth noting that it is found that 6 At sites with highly overlapping peaks, Ythdf2 binds to transcription factor mRNAs (e.g., Tal1 and Gata2 mRNAs) ( Figure 3E ; Figure 5E and Table S2, Ythdf2-targeted mRNAs from three irCLIP-seq replicates). LSK Flk2 in wt and Ythdf2 KO mice - No significant changes were observed in the total RNA quality of the cells. 6 A modification constitutes only 0.1-0.4% of adenosine nucleotides in mammalian cells, but Ythdf2 KO was found to cause BM Lin - m in total RNA of cells 6 A content increased, indicating that Ythdf2 specifically regulates m 6 The stability of A-labeled mRNA ( Figure 12A and 12B Consistently, qPCR analysis of total mRNA revealed that Tal1, Gata2, Runx1, and Stat5a (whose mRNAs have been shown to be expressed by mRNAs) were down-regulated in Ythdf2 KO LSK cells compared with wt controls. 6 A modification) levels increased ( Figure 3F Single-cell immunofluorescence staining and intracellular flow cytometry further showed that Ythdf2 KO HSPCs exhibited mRNAs involved in stem cell self-renewal. 6The intensities of A-marked transcription factors (such as TAL1, GATA2, RUNX1, and STAT5) were significantly increased, indicating the inhibitory role of Ythdf2 in HSC self-renewal ( Figure 3G ; Figure 12C ). Previous studies have shown that Ythdf2 regulates RNA metabolism by localizing bound mRNA to mRNA decay sites (Wang et al. Nature, 505: 117-120, 2014). To further explore the mechanism by which Ythdf2 regulates HSC self-renewal, fluorescence in situ hybridization (FISH) was performed on Tal1 mRNA, and Ythdf2 and Dcp1a, markers of mRNA decay sites (Sheth et al. Science, 30: 805-808, 2003; Kedersha et al. Methods in Enzymology, 431: 61-81, 2007), were fluorescently immunostained, and their relative spatial distribution was analyzed in sorted wt and Ythdf2 KO HSPCs. Co-localization of Tal1 mRNA, Dcp1a, and Ythdf2 was observed in wt cells, but was significantly reduced in Ythdf2 KO controls ( Figure 3H and 3I ). In addition, similar observations were confirmed by co-staining of Gata2 mRNA FISH with Ythdf2 and Dcp1a in wt or Ythdf2 KO HSPCs ( Figure 12D and 12E To determine whether increased expression of transcription factors such as Tal1 contributes to HSC expansion in Ythdf2 KO mice, rescue experiments were performed using short hairpin (sh)RNA-mediated knockdown (KD) of Tal1 in wt and Ythdf2 KOLSK cells followed by transplantation into lethally irradiated recipients. As previously reported, depletion of Tal1 in HSPCs significantly impaired the reconstitution capacity of wt cells and also rescued the increased engraftment of Ythdf2 KO cells ( Figure 12F Collectively, these data suggest that Ythdf2 regulates HSC self-renewal by enabling the degradation of mRNAs encoding transcription factors essential for stem cell renewal.

[0269] Example A-4

[0270] By m 6 A-seq and RNA-seq analysis of the role of Ythdf2 in human UCB HSPCs.

[0271] The limited number of HSCs in a single human umbilical cord blood unit has been a barrier to clinical applications such as HSC transplantation (Walasek et al., Annals of the New York Academy of Sciences, 1266:138-150, 2012). The observation that Ythdf2 KO results in an increase in phenotypic and functional mouse HSCs prompted the test of whether YTHDF2 knockdown (KD) can promote the expansion of human UCB HSCs. First, CD34 isolated from 3 separate hUCB samples was used to investigate the effect of YTHDF2 knockdown on the HSCs. + Cellular m 6 A-seq( Figure 13A As expected (about 90%), m 6 A modification occurs mainly in mRNA (about 95%), preferentially in the mRNA ORF region, 3'UTR and near the termination codon ( Figure 4A and 4B ; Figure 13B ). The m in mouse and hUCBHSPCs were compared. 6 A landscape, 2,239 genes were found to be commonly m 6 A mark ( Figure 4C ). These are usually 6 A-labeled transcripts are enriched in genes related to hematopoiesis and stem cell maintenance ( Figure 4D ). Due to the mRNA of the transcription factor responsible for mouse HSC self-renewal 6 A marker enrichment, we then characterized hUCB CD34 by performing GO term analysis. + m 6 A-labeled transcription factor transcripts. Among 722 identified m 6 Among the A-labeled transcription factor mRNAs, the main GO terms were related to cell fate determination and stem cell maintenance ( Figure 13C For example, it has been reported that HOXB4 overexpression in hUCBCD34 cells is involved in the in vitro expansion of human and mouse HSCs (Amsellem et al. Nature Medicine, 9:1423-1427, 2003; Antonchuck et al. Cell, 109:39-45, 2002). + cells are m 6 A mark ( Figure 4E Other transcription factors required for HSC self-renewal and crucial for the induction of HSC from other cell types (Ebina et al. The EMBO Journal, 34: 694-709, 2015; Galan-Caridad et al. Cell, 129: 345-357, 2007) (e.g., Zfx, RUNX1, and FOSB) are expressed in hUCB CD34+ In cells, there is also m 6 A-labeled ( Figure 13F and 13G , see also Supplementary Table S3 of the article “Supression of m6AReader Ythdf2Prmotd Hematopoeitic Stem Cell Expansion” by Li et al., Cell Research 28, 904-917 (2018), which is incorporated herein by reference in its entirety (including its supplementary tables), for human UCB CD34 from a separate sample + HSPC 6 To further analyze the role of YTHDF2 in hUCB HSPCs, control or YTHDF2 KD hUCB CD34 + Cells were subjected to RNA-seq ( Figure 4F ; Figure 13D and 13E ). Remarkably, by m 6 A-marked transcripts, including HOXB4 and other HSC self-renewal-related transcription factors, showed a significant increase in input mRNA sequencing fragments in YTHDF2 KD cells compared with controls, whereas for non-m 6 There was no significant change in the genes marked by A ( Figures 4G to 4I ; 13F and 13G). These results support a role for YTHDF2 in regulating hUCB HSC self-renewal through RNA degradation.

[0272] Example A-5

[0273] Expansion of hUCB HSCs by YTHDF2 KD.

[0274] To further explore whether YTHDF2 inhibition can expand human HSCs, short hairpin (sh)RNA-induced YTHDF2 KD was performed in hUCB HSPCs as described above. Figure 4F After 7 days of in vitro culture, lentiviral knockdown of YTHDF2 resulted in a decrease in CD34 + CD38 - CD45RA - EPCR + The frequency and absolute number of phenotypic HSCs increased by an average of 14.3-fold and 13.6-fold, respectively, and resulted in a 5.1-fold increase in CFUs, reflecting the higher expression levels of key transcription factors for hematopoiesis, such as TAL1, in YTHDF2 KD hUCB cells. Figures 5A to 5D ; Figure 14A Interestingly, the apoptosis rate in YTHDF2 KD hUCB HSPCs was significantly reduced compared with control cells, similar to the trend observed in HSCs from Ythdf2 KO mice ( Figure 5E ).and, Figure 5F The results showed that Ythdf2 knockdown (KD) HSCs showed up to a 10-fold increase compared to control HSCs 10 days after transduction. Next, the effect of overexpression (OE) of YTHDF2 on HSPC function was explored. Overexpression of YTHDF2 reduced the clonal potential of hUCB HSPCs by 2.2-fold, indicating that YTHDF2 negatively regulates HSC maintenance in vitro. Figure 14B and 14C ).

[0275] To determine whether YTHDF2 KD could expand human HSCs in vivo, we transplanted CD34 HSCs from hUCB infected with control and YTHDF2 shRNA at day 4 post-infection. + GFP sorting in cells + Cells were subjected to LDA ( Figure 6A ). Ten weeks after transplantation, we analyzed the expression of Il2rg in recipient NOD / SCID cells. null BM cells from (NSG) mice were harvested and functional HSC frequencies were measured after in vivo expansion. Notably, YTHDF2 KD cells had a significantly higher number of human hematopoietic cells (hCD45 + GFP + ) engraftment increased 9-fold, while the proportion of each lineage remained unchanged ( Figure 6B and 6C H; Figure 15A and 15B YTHDF2 KD significantly increased the percentages of myeloid cells, megakaryocytes, and erythrocytes in the BM of primary recipients ( Figure 15C Thus, it was found that the frequency of HSCs in YTHDF2 KD cells increased 4.4-fold compared to control cells ( Figure 6D This confirmed that YTHDF2 KD hUCB cells have the long-term capacity to be reconstituted and undergo self-renewal; 12 weeks after BM transplantation from primary recipients into sublethally irradiated secondary NSG recipient mice, human hematopoietic cell chimerism was higher in the BM of the YTHDF2 KD group compared with the control group ( Figure 6E 、 6F ; Figure 15D ). CRU from YTHDF2 KD cells showed an 8-fold increase relative to control cells ( Figure 6G These data indicate that YTHDF2 KD significantly promotes the phenotypic and functional expansion of hUCB HSCs in vitro.

[0276] Example A-6

[0277] The examples herein demonstrate that conditional deletion of Ythdf2 (m 6 A) resulted in the expansion of phenotypic and functional HSCs without lineage bias. To investigate whether there is a concomitant increase in mesenchymal stem cells in vivo, Ytdhf2 f / f Mice were crossed with Mx1-Cre mice to conditionally delete Ythdf2 expression from mesenchymal cells. KO Bone marrow cells were isolated from wild-type littermates. Single-cell suspensions of total bone marrow cells were immunostained with flow cytometry antibodies. Hematopoietic cells (CD45, Ter119) and endothelial cells (CD31) were excluded using specific markers. Mesenchymal stem cells present within total bone marrow stromal cells were further purified using antibodies containing N-cadherin and CD105. + and CD105 + Conditional deletion of Ythdf2 leads to a 3.6-fold increase in the frequency of mesenchymal stem cells ( Figure 16 Our study clearly demonstrated that loss of Ythdf2 can expand bone marrow mesenchymal stem cells in vivo.

[0278] discuss

[0279] Although recent studies have explored the 6A modified biological function (Zheng et al. Molecular Cell, 49: 18-29, 2013; Zhou et al. Nature, 526: 591-594, 2015; Alarcon et al. Cell, 162: 1299-1308, 2015; Zhang et al. Cancer Cell, 31: 591-606e596, 2017; Lence et al. Nature, 540: 242-247, 2016; Haussmann et al. Nature, 540: 301-302, 2016; Chen et al. Cell Stem Cell, 16: 289-301, 2015; Alarcon et al. Nature, 519: 482-485, 2015; Xiao et al. Molecular Cell, 61: 507-519, 2016; Wojtas et al. Molecular Cell, 61: 507-519, 2016). Cell, 68:374-387e312, 2017; Ivanovna et al. Molecular Cell, 67:1059-1067e1054, 2017; Fustin et al. Cell, 155:793-806, 2013; Slobodin et al. Cell, 169:326-337e312, 2017; Schwartz et al. Cell, 159:148-162, 2014; Pendleton et al. Cell, 169:824-835e814, 2017; Shi et al. Cell Research, 27:315-328, 2017; Huang et al. Nature Cell Biology, 20: 285-295, 2018; Bertero et al. Nature, 2018; Liu et al. Nature, 518: 560-564, 2015), the embodiments of this invention are to convert the m 6 A modification is coupled to the degradation of mRNAs encoding key self-renewal transcription factors, identifying Ythdf2 as a crucial regulator of human and mouse HSC self-renewal. Ythdf2 inhibition in mouse HSPCs and hUCB HSCs leads to increased expression of multiple key TFs essential for self-renewal, promoting the ex vivo expansion of phenotypic and functional HSCs without significant lineage bias or leukemic potential. Furthermore, the stem cell niche may contribute in part to Ythdf2 inhibition-mediated mouse HSC expansion, as Mx1-cre can be activated in mesenchymal stromal cells. It will be fascinating to investigate the function of Ythdf2 on mesenchymal stem cells (MSCs) and how Ythdf2 inhibition in HSCs and MSCs synergizes HSC expansion in vivo.

[0280] Given that m6 The A-script complexes Mettl3 and Mettl14 have extensive and complex effects on mRNA splicing, translation, and pri-miRNA processing (Barbieri et al. Nature, 2017; Alarcon et al. Nature, 519:482-485, 2015; Liu et al. Nature, 518:560-564, 2015). Depletion of Mettl3 or Mettl14 leads to different outcomes in normal stem cells and leukemia. Recent studies have shown that Mettl3 and Mettl14 play a crucial role in the development of leukemia and the maintenance of leukemia stem cells (Vu et al. Nature Medicine, 2017; Barbieri et al. Nature, 2017; Weng et al. Cell Stem Cell, 22:191-205e199, 2018). In contrast, Ythdf2 is believed to be primarily involved in m 6 A-mediated mRNA decay (Batista et al. Cell Stem Cell, 15:707-719, 2014; Yoon et al. Cell, 2017; Zhang et al. Nature, 549:273-276, 2017; Wang et al. Nature, 505:117-120, 2014). According to certain embodiments herein, it is believed that manipulation of Ythdf2 can prolong the expression of specific mRNAs encoding TFs that are essential for stem cell self-renewal. 6 The half-life of A-labeled mRNA is reduced without affecting other aspects of mRNA processing. The examples herein show that Ythdf2 depletion in HSCs neither reverses lineage commitment nor induces hematological malignancies, thereby reducing the risk of leukemia development in the presence of HSC expansion. In addition, stem cell self-renewal is a complex process involving cell division, survival, prevention of differentiation, and maintenance of stemness. The observation that Ythdf2-deficient HSCs exhibit a lower rate of apoptosis suggests that embodiments of the methods herein also benefit another characteristic of stem cell self-renewal.

[0281] The main limitation of using hUCB HSC transplantation is the insufficient number of HSCs in one hUCB unit. Although previous studies have shown that Dlk1, SR1, Musashi2, and UM171 can amplify hUCB HSCs by targeting Notch, AHR signaling, or other unknown pathways (Boitano et al. Science, 329: 1345-1348, 2010; Fares et al. Science, 345: 1509-1512, 2014; Rentas et al. Nature, 532: 508-511, 2016; Chou et al. Experimental Hematology I, 41: 479-490e474, 2013). Therefore, the embodiments herein provide novel and effective methods to target multiple key TFs that are crucial for HSC self-renewal and enhance the expansion of HSCs. For example, reducing the level and function of Ythdf2 during in vitro culture by small chemicals or AAV-mediated KD can restore the level and function of Ythdf2 after transplantation in vivo, thereby not affecting the normal maintenance and function of HSCs in human patients. In addition, in certain embodiments, the methods described herein can be combined with other methods to promote the expansion of not only human HSCs, but also other stem cells, thereby providing methods for stem cell-based therapies.

[0282] Example B

[0283] The following protocol was used in the "B" examples below.

[0284] animal. C57BL / 6-Gt(ROSA)26Sortm1(HBEGF)Awai / J(iDTR), B6.Cg-Gt(ROSA)26Sortm14(CAG-tdToma to)Hze / J(R26RtdT)、Tg(Cspg4-DsRed.T1)1Akik / J、Cxcl12tm2.1Sjm / J、Kitltm2.1Sjm / J(SCF f / f )、Cxcl12tm1.1Sjm / J(CXCL12 f / f ) mice were obtained from Jackson Laboratory. T and N-cad-TdT mice were generated by Applied StemCell, Inc. To induce N-cad-CreER T R26-tdT mice were injected intraperitoneally with 2 mg of tamoxifen (Sigma) for 3 days. TR26-tdT, a single dose of 1.5 mg TMX was injected intraperitoneally (IP) into pregnant females at E12.5. Cesarean sections were performed at E19.5, and newborn mice were transferred to foster mice. T ; Induction of N-Cad in iDTR mice + Cell ablation was performed by intraperitoneal injection of DT (Sigma) at a dose of 50 ng per gram of body weight every other day as indicated. 5FU (Sigma-Aldrich) was injected once in the tail vein at 150 μg per gram of body weight. After injection of 5FU, mice were analyzed as described in the text. All mouse lines used in this study had a C57BL / 6J genetic background. Animals were randomly included in the experiments based on genotyping results. Animal experiments were performed in a blinded manner to the researchers. The number of animals used in each experiment is indicated in the figure legends. All mice used in this study were housed in the animal facility of the Stowers Institute for Medical Research (SIMR) and handled according to SIMR and National Institutes of Health (NIH) guidelines. All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of SIMR.

[0285] Flow cytometry. For phenotypic analysis, hematopoietic cells were harvested from bone marrow (femur and tibia). Red blood cells were lysed using 0.16 M ammonium chloride solution. For cell surface typing, a lineage cocktail (Lin, phycoerythrin (PE)-Cy5) was used, including anti-CD3 (145-2C11), anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-Mac-1 (M1 / 70), anti-Gr1 (RB6-8C5), anti-B220 (RA3-6B2), anti-IgM (II / 41), and anti-TER119 (TER-119) (100 ng antibody cocktail per million bone marrow cells, eBioscience). Monoclonal antibodies against SCA1 (D7, eBioscience), c-KIT (2B8, eBioscience), FLK2 (A2F10, eBioscience), CD34 (RAM34, eBioscience), CD48 (HM48-1, eBioscience), CD150 (TC15-12F12.2, BioLegend), and CD49b (HMα2, Biolegend) were also used as indicated (all used at 50 ng per million bone marrow cells). For lineage analysis of peripheral blood, monoclonal antibodies against CD45.1 (A20, eBioscience), CD45.2 (104, eBioscience), CD3, B220, Mac-1, and Gr1 were used. Dead cells were excluded using 7-aminoactinomycin D (7-AAD) (A1310, Life technologies). For stromal niche cell analysis, CD45 (30-F11, eBioscience), CD31 (390, eBioscience), PDGFRα-biotin (APB5, eBioscience), LepR-Bio (R&D), and CD51 (clone RM7-V, Biolegend) were used. Samples stained with biotin-conjugated antibodies were washed with staining medium and then incubated with Streptavidin Brilliant Violet 421™ (Biolegend, 1:500). Cell sorting and analysis were performed using a MoFlo (Dako), InFlux cell sorter (BD Biosciences), MACSQuant (Miltenyi Biotec), or CyAn ADP (Dako) instrument. Data analysis was performed using FlowJo software.

[0286] Whole-embedded sternal HSC immunostaining. The sternum was collected and cut into 3-4 fragments with a scalpel blade. The fragments were cut sagittally to expose the bone marrow cavity and fixed in 4% PFA. The tissue was blocked / permeabilized in PBS containing 20% ​​normal goat serum and 0.5% Triton X-100 and stained with primary antibodies for 3 days. The tissue was incubated with secondary antibodies for 2 hours (Bruns et al., 2014; Kunisaki et al., 2013). Fluorescence imaging was performed on a spinning disk confocal microscope (UltraVIEW; PerkinElmer) (including an inverted microscope (Axiovert 200M; Carl Zeiss Microimaging, Jena, Germany)) connected to a spinning disk confocal system (CSU-X1; Yokogawa Corporation of America) and equipped with Volocity acquisition software (PerkinElmer) and an Orca-R2 camera (Hamamatsu) with a 20x / 0.8 Plan-Apochromat objective lens (Carl Zeiss). The image is collected as a series of optical profiles (section) with a step length of 4 μm. Images are collected in a tile mode (10% overlap) sufficient to cover the entire sample. Channels are collected sequentially. (Blue dye) uses 405nm light (50mW diode laser, OEM) to excite, (Red dye) uses 561nm light (50mW solid-state laser, OEM) to excite, and each uses a multi-bandpass emission filter of 415nm-775nm 580nm-650nm band to collect. (Green dye) uses 488nm light (50mW solid-state laser, OEM) to excite and uses a multi-bandpass emission filter of 500nm-550nm to collect, (Far red dye) uses 640nm light (50mW solid-state laser, OEM) to excite, and uses a multi-bandpass emission filter of 455nm-515nm and 660nm-750nm to collect. Adjust exposure time and laser power to compensate for changes in staining.

[0287] Second harmonic generation (SHG) imaging was performed immediately after fluorescence imaging. SHG images were collected on an LSM-780 laser scanning confocal microscope (Carl Zeiss) equipped with a QUASAR detection unit, a 10x 0.45 Plan-Apochromat objective lens (Carl Zeiss) and Zen 2012 acquisition software (v8.1.3, Carl Zeiss). Images were collected as a series of optical profiles with a step size of 8 μm and a pixel size (1.32 μm / pixel) that was an integer multiple of four times the fluorescence pixel size. SHG images were taken with a laser wavelength of 900 nm and collected at 371-420 nm. As a reference for image comparison, images of Cd150-PE were collected at 566-735 nm using the 561 nm line of a DPSS laser (Melles Griot) and simultaneously with the SHG images. Images were collected in a tile mode (no overlap) sufficient to cover the entire sample. The tiles were stitched into a complete 3D image using Zen software.

[0288] Images were analyzed using Fiji software (1.51 g National Institutes of Health). To align the SHG and fluorescence images, the background of the fluorescence image was first subtracted, and then the image tiles were stitched into a complete 3D image using the Grid / Collection stitching plug-in (see http: / / bioinformatics.oxfordjournals.org / content / 25 / 11 / 1463.abstract). Given that transferring the sample from the fluorescence microscope to the SHG microscope involves a small amount of sample rotation, it was necessary to realign the SHG and fluorescence images in three dimensions. The alignment of the SHG and fluorescence image sets was performed using a custom plug-in available at http: / / research.stowers.org / imagejplugins. First, a minimum of eight common landmarks were identified by visual inspection of the SHG and fluorescence datasets. Next, the Kabsch algorithm was used to find the optimal scaling rotation to convert the fluorescence coordinates to SHG image coordinates. Finally, each 3D voxel in the fluorescence image was transformed to the corresponding SHG position, and trilinear interpolation was used to find the SHG intensity at that position to create a realigned composite image.

[0289] Image analysis was performed by researchers who were unfamiliar with the study hypothesis. HSCs were identified visually. If the shape of the cell could not be identified with the naked eye, or if the shape of the cell formed a dark area in the field of view of the positive signal, the cell was considered negative for staining. Distance measurements of niche components were performed using Fiji and Microsoft Excel software. The position of the HSC and the nearest point of each of the three niche components were marked using point ROIs in Fiji, and the positions were transferred to Excel, and the distances in 3D were calculated using the 3D Pythagorean theorem. To calculate the distances of randomly distributed HSCs, a custom plug-in in FIJI was used to generate random point ROIs in the same images analyzed for the observed HSCs. If the randomly generated points appeared in a reasonable HSC position (assessed by the presence of Lin+ cells in the surrounding area), they were considered to be simulated HSCs. Niche distance measurements were then performed on the simulated HSCs in the same manner as for the observed HSCs.

[0290] The statistical significance of differences in distance distribution was assessed by Kolmogorov-Smirnov analysis using Origin software. The statistical significance of changes in the percentage of HSCs at 5 μm was assessed using the Student's T test in Microsoft Excel. Changes were considered significant if P < 0.05.

[0291] The final images shown in the accompanying figures are maximum projections from which the background has been subtracted and contrast adjusted for clarity.

[0292] Transplantation and repopulation assay. 100 sorted pHSC or rHSC cells were mixed with 1.0 × 10 5 CD45.1 rescued bone marrow cells were transplanted into lethally irradiated (10 Gy) CD45.1 recipients. T ; 2.0×10 5 CD45.2BM cells and 2.0×10 5 CD45.1 rescued bone marrow cells were transplanted into lethally irradiated (10 Gy) CD45.1 recipients. Peripheral blood was collected from the submandibular vein every 4 weeks after transplantation. Hematopoietic repopulation was measured from donor-derived blood cells (CD45.2).

[0293] RNA sequencing and analysis. cDNA was generated from 1000 purified cells using the SMARTer Ultra Low Input RNA Kit (Clonetech), and libraries were generated using the Nextera XT DNA Library Preparation Kit (Illumina). 50-bp single-read reads were then sequenced on an Illumina HiSeq2500. Raw reads were demultiplexed into Fastq format using Illumina bcl2fastq2 v2.18, allowing for up to one mismatch. Reads were aligned to the UCSC genome mm10 using TopHat v2.0.13 with default parameters. FPKM values ​​were generated using Cufflinks v2.2.1 with the command "-u –max-bundle-frags 100000000". Read counts were generated using HTSeq-count with the command "-m intersection-nonempty". Three or four replicates were sequenced for each population. Data are available at NCBI GEO: GSE104887.

[0294] CFU-F assay and in vitro differentiation. Cells were sorted directly into 96-well plates. Cultures were incubated at 37°C in a humidified atmosphere of 5% O2 and 10% CO2 for 7-10 days. TM Colonies were stained with Green CMFDA (Life Technologies) and images were acquired using a Celigo Imaging Cytometer (Nexcelom). For in vitro differentiation, clonally expanded Ncad-CreER was isolated from CFU-F cultures by digestion with 0.25% trypsin / EDTA. T Tomato powered + BM / bone stromal cells were divided into three equal parts and seeded into separate cultures that allowed differentiation: StemPro Osteogenic Kit Gibco A10072-01, Adipogenic Kit A10070-01, and Chondrogenic Differentiation Kit A10071-01. Osteogenic differentiation was assessed using VECTOR Red alkaline phosphatase; adipogenic differentiation was detected using Oil Red O (Sigma); and chondrogenic differentiation was detected using Toluidine Blue (Sigma, 0.1 g T Blue / 100 mL distilled water).

[0295] Bone sectioning, immunostaining, and imaging.

[0296] Freshly isolated femurs were fixed overnight in 4% paraformaldehyde and then decalcified in 10% EDTA for 1 to 3 days. For paraffin sections, bone samples were processed using a Sakura Tissue Tek VIP 5 tissue processor (Sakura America, Torrance, CA) and paraffin sections were cut into 5 μm thickness. Sections were dewaxed with xylene and then stained with Alcian blue / hematoxylin / orange G. For frozen sections, bone samples were processed using a CryoJane tape transfer system. Sections were fixed using a PowerBlock 3000 microscope. TM The cells were blocked with a universal blocking agent for 30 minutes to 1 hour, and then stained overnight with rabbit anti-aggregate (Millipore, 1:300), rabbit anti-perilipin (Cell Signaling, 1:300) and goat anti-osteopontin (R&D, 1:300). Donkey anti-goat Alexa Fluor 488 and donkey anti-goat Alexa Fluor 647 were used as secondary antibodies (all from Invitrogen, 1:300). Antibodies were diluted with antibody diluent (Invitrogen 00-3218). Slides were fixed with FLUORO-GEL (Electron Microscopy Science 1798510) and images were collected with an Olympus slide scanner.

[0297] Femoral groove surgery. Mice were anesthetized with 2.5% isoflurane and buprenorphine was administered for analgesia. The skin of the right leg was shaved and scrubbed with alcohol and iodine. A small incision was made in the skin on the lateral side of the knee joint. After sliding the skin medially for visualization, an internal incision was made on the medial side of the kneecap, extending to the quadriceps and along the patellar tendon to release the tissue. The patella was subluxated laterally and the distal femur was exposed. A microsaw was used to perforate the subchondral bone to penetrate the articular cartilage of the knee joint. The extensor structures (quadriceps, patellar tendon and patella) were restored to their original anatomical position. The internal incision was closed with absorbable sutures and the skin was closed with non-absorbable sutures.

[0298] statistics.

[0299] Values ​​are shown as mean ± sem. All statistical analyses were generated using GraphPad Prism 5 (GraphPad Software). Student's t-test was used for comparisons between two groups. Statistical significance was defined as p < 0.05.

[0300] Example B-1

[0301] Functionally distinct reserve and primed HSCs exist in mouse bone marrow. To explore the reserve HSC (hereafter referred to as rHSC) subpopulation, the cell surface marker CD49b (integrin α2) was adapted. CD49b can distinguish intermediate HSCs from permanent long-term HSCs (LT-HSCs) (Benveniste et al., 2010; Qian et al., 2015; Wagers and Weissman, 2005; Yang et al., 2005). Interestingly, CD48 – CD49b – The subpopulation is only present in conventional LT-HSCs (CD34 – Flk2 – Lineage – Sca-1 + c-Kit + (LSK) cells), while in short-term HSCs (ST-HSC; CD34 + FLK2 - LSK) or multipotent progenitor cells (MPP; CD34 + FLK2 + It is not present in LSK. – CD49b – The LT-HSC subpopulation is enriched for rHSC, while CD48 – CD49b + The LT-HSC subpopulation is enriched with sensitized HSC (hereinafter referred to as pHSC) ( Figure 17A ) and were tested using a repopulation assay. Both rHSCs and pHSCs were found to support hematopoiesis in lethally irradiated mice for up to 40 weeks after transplantation without significant differences ( Figure 17B ), which is consistent with previous reports (Benveniste et al., 2010). However, compared with transplanted rHSCs, transplanted pHSCs were very inefficient in generating rHSCs (95.4% reduction) as well as ST-HSCs and MPPs in the recipient (about 78% reduction for both populations), indicating that rHSCs are superior to pHSCs in terms of hierarchy ( Figure 17C Using the Scl-tTA-induced H2B-GFP tag retention model, we found that rHSCs significantly enriched more H2B-GFP compared to pHSCs. high cells (P = 0.0039), indicating that rHSCs have a slower cell cycle than pHSCs ( Figure 17DAt the molecular level, rHSCs were found to have higher expression of Gadd45g, Cdkn1c (encoding p57), and Foxo1, all involved in maintaining the G0 phase of HSCs, and lower expression of cell cycle activators such as Myc, Pcna, Ccng2, and Cdk4. There was no difference in Ki67 expression between rHSCs and pHSCs, indicating that Ki67 expression alone is not sufficient to distinguish the two HSC subpopulations ( Figure 17E ).

[0302] Because the functional definition of rHSCs is drug resistance, rHSCs or pHSCs were transplanted into recipient mice, and the mice were challenged with 5FU 4 weeks after transplantation. Figure 17F As shown in Figure 3, rHSCs were insensitive to 5FU treatment; however, pHSCs significantly reduced their reconstitutive capacity (44% reduction 20 weeks after transplantation). – CD49b – LT-HSCs are indeed enriched for rHSCs that are resistant to chemotherapy, and CD48 – CD49b + LT-HSCs enriched for chemotherapy-sensitive pHSCs; moreover, in transplantation assays, the former gave rise to the latter, but not vice versa.

[0303] The direct consequences of acute 5FU challenge on rHSCs and pHSCs were further analyzed. Figure 17G As shown in Figure 3, 3 days after 5FU, approximately 92% of pHSCs were eliminated and only rHSCs survived, suggesting that rHSCs must have specifically turned on their DNA repair systems to be able to overcome chemotherapy stress. To test this hypothesis, the transcriptome profiles of DNA damage response genes in rHSCs, pHSCs, and rHSCs after 5FU treatment were analyzed. It was observed that during homeostasis, rHSCs maintained lower expression of genes involved in the DNA damage repair system compared to pHSCs ( Figure 17H ), but most DNA repair pathways such as DNA mismatch repair (MMR), nucleotide excision repair (NER), base excision repair (BER), and homologous recombination (HR) were significantly activated in rHSCs under 5FU challenge ( Figure 17I In addition, in parallel, a large number of stress response genes mainly belonging to the Hsp90 and Hsp70 families (Rodina et al., 2016) were also upregulated (1.8 ± 0.17-fold and 1.4 ± 0.1-fold, respectively) ( Figure 17J ), which partly explains how rHSCs survive chemotherapeutic stress and reconstitute the hematopoietic system.

[0304] In conclusion, we functionally demonstrated the coexistence of pHSCs and rHSCs in the BM. Even in their quiescent state and with activated DNA repair pathways, pHSCs remain sensitive to chemotherapy, whereas rHSCs activate their DNA damage repair and stress response genes to survive chemotherapeutic stress and generate pHSCs. Thus, rHSCs play a crucial role in supporting hematopoietic regeneration under severe stress.

[0305] Example B-2

[0306] Drug-resistant rHSCs are mainly located in the endosteal region of the bone marrow

[0307] We further examined whether exogenous mechanisms from the BM niche contribute to the maintenance of rHSCs during homeostasis and under chemotherapeutic stress. To this end, whole-mount HSC staining was performed, which simultaneously examined the relative distribution of rHSCs and pHSCs within bone (by second harmonic generation (SHG)), megakaryocytes (MKs), or blood vessels within a bone cavity approximately 75 μm thick ( Figure 18A -B). Our quantitative data showed that 43.4% of rHSCs and 31.0% of pHSCs were located within a distance of 10 μm from blood vessels, and 22.6% of rHSCs and 24.6% of pHSCs were located within a distance of 10 μm from MKs ( Figure 18C -D), consistent with previous reports that the majority of HSC populations were located in the perivascular and sinusoidal regions (Acar et al., 2015; Bruns et al., 2014; Chen et al., 2016; Zhao et al., 2014). Interestingly, it was noted that 16.4% of rHSCs were located within 10 μm of the talar surface, compared to only 3.69% of pHSCs ( Figure 2E These data indicate that both rHSCs and pHSCs were distributed in blood vessels and MKs without bias, but rHSCs were located significantly closer to the endosteal bone surface compared with pHSCs (P = 0.00182).

[0308] To test whether the endosteal region retains rHSCs during chemotherapeutic stress, the distribution of rHSCs was examined on day 3 after 5FU treatment when pHSCs were depleted ( Figure 18B Interestingly, we found that upon acute 5FU stress, approximately 55% of surviving rHSCs were retained by the endosteal niche, which is approximately a 3.5-fold enrichment compared to steady-state ( Figure 18E However, no significant differences were observed in the frequencies of surviving rHSCs near blood vessels or MKs ( Figure 18C -D). Consistently, as Figure 17GAs shown, only 4.24% of pHSCs were observed to survive acute 5FU stress. Next, the dynamic process of BM injury and subsequent recovery after 5FU stress was investigated by examining BrdU-labeled surviving and proliferating cells. It was noted that on day 2 after 5FU treatment, a large loss of BrdU-labeled cells was observed, indicating active cell apoptosis induced by 5FU. On day 3 after 5FU treatment, surviving BrdU cells were observed. + Cells (mostly single cells) were detected primarily near bone lining cells in the endosteal region ( Figure 24A -D). On day 3.5, paired BrdU was observed + Cells appeared on the endosteal surface, indicating activation and division of surviving cells. Starting from days 4 and 5 and continuing, the number of proliferating cells gradually increased, and these cells were often detected as clusters near blood vessels or potential adipocyte structures (approximately 55% on day 4 and approximately 65% ​​on day 6) ( Figure 24E -I). This observation suggests that the bone surface is the niche where cells initially survive after 5FU treatment. These cells are then activated and produce daughter cells, which primarily proliferate in blood vessels or near megakaryocytes (Zhao et al., 2014). It was further confirmed that surviving 5FU-rHSCs were indeed often detected as single cells adjacent to the bone surface, and that proliferating HSCs were often associated with MKs or near blood vessels. Surviving rHSCs (green, CD150) on day 3 after 5FU treatment + Lin - CD49b-) are usually detected as single cells adjacent to the bone surface (white, SHG), and proliferating HSCs are often associated with MKs (CD150 + Lin+) or near blood vessels (red, CD31+) ( Figure 29 ).

[0309] N-cad has been found on the bone surface + Osteoblast precursor cells are resistant, and Osx has been found + Osteoblasts are sensitive to 5FU treatment, and N-cad + Stromal cells produce Osx during recovery after 5FU treatment + A recent study showed that 1 day after irradiation, LepR in the central bone marrow was expressed due to cell death (Sugimura et al., 2012). + Stromal cells decreased dramatically (Zhou et al., 2017). To track the early changes in the vascular and endosteal niches, apoptosis assays were performed on day 1 after 5FU treatment. + VE-cadherin + The cells increased significantly ( Figure 18F), which is consistent with the previously reported destruction of vascular structure after 5FU (Dominici et al., 2009; Sugimura et al., 2012). To study the endosteal niche, the N-cad-tdTomato (N-cad-TdT) mouse line was established, in which Tomato + Cells expressed N-cad in both the central bone marrow and the bone surface ( Figure 24J ). Note that apoptotic N-Cad-driven Tomato + The number of cells in the endosteal region increased significantly, and the number of Tomato cells in the endosteal region increased significantly after 5FU treatment. + The cells remain stable ( Figure 18G On day 3 after 5FU treatment, when hematopoietic regeneration begins, N-Cad-driven Tomato + The cells increased by 2.2 times and 1.7 times ( Figure 18H , Figure 24K ). CD31 + Vascular cells also increased in number but showed dilated and damaged structures ( Figure 18I Taken together, previous reports and this data suggest that blood vessels and associated stromal cells (including LepR + and N-cad + cells) were immediately damaged and sensitive to 5FU stress, while N-cad + Endosteal stromal cells remained stable.

[0310] These data partially explain previous findings that most HSCs are primarily located in perivascular and sinusoidal regions (Acar et al., 2015; Chen et al., 2016; Kunisaki et al., 2013). pHSCs, which comprise 51.3% of the quiescent HSC population, are located near perivascular and sinusoidal regions during steady-state. However, under stress, rHSCs move closer to the bone surface and survive chemotherapy. Taken together, these data suggest that the endosteal niche plays a key role in protecting rHSCs against chemotherapy.

[0311] Example B-3

[0312] N-Cad + Niche cells maintain functional HSCs, including rHSCs, in the bone marrow

[0313] Although N-cad + Stromal cells were the first HSC niche cells to be identified, and although N-cad + Stromal cells are resistant to chemotherapy and Osx +Osteoblasts are sensitive to chemotherapy, but N-cad is still deficient due to the lack of appropriate genetic mouse lines. + Direct evidence of functional support for HSCs. According to aspects of this paper, N-cad-CreER was generated T Tie( Figure 25A ) and displays N-cad + Stromal cells produce Col2.3-GFP + Osteoblasts and perivascular cells ( Figure 25B ). Previous studies have shown that depletion of mature Col2.3-GFP + Osteoblasts do not affect the overall engraftment of BM cells (Ding et al., 2012; Greenbaum et al., 2013), but only lead to impaired regenerative capacity of the LT-HSC subset (Bowers et al., 2015). + Stromal cells developmentally continue to develop into Osx + osteoprogenitor cells, which further generate mature Col2.3 + osteoblasts, but N-cad + The functional contribution of stromal cells to HSC maintenance is still unclear. + The HSC niche of the cell generates N-cad-CreER T Inducible DTR (encoding diphtheria toxin receptor) line (N-cad-CreER T ; iDTR), where N-cad + Niche cells are sensitive to diphtheria toxin (DT). T iDTR mice were given three tamoxifen (TMX) injections followed by intraperitoneal injections of DT (once every other day) and analyzed on the day after the last injection ( Figure 19A ). N-cad-CreER treated with DT at the same time T Compared with R26-tdT mice, N-cad-CreER T N-cad was observed in ;iDTR;R26-tdT + Effective ablation of stromal cells ( Figure 19B ). Further examination of N-cad + Does cell ablation affect HSCs in vivo? + After 14 h of BM, no significant changes in cellularity were observed compared with controls ( Figure 19C However, the number of HSCs was significantly reduced: rHSCs (reduced by 65.0%), pHSCs (reduced by 60.0%), ST-HSCs (reduced by 59.6%), and MPPs (reduced by 29.3%) ( Figure 19D ). This indicates that N-cad + Niche cells contribute to the maintenance of the most primitive (including reserve) HSCs.

[0314] Transplantation assays were also performed to test N-cad + The number of functional HSCs in stromal cell ablated mice has been found to be increased by N-cad + Bone marrow cells from stromal cell-ablated mice gave significantly lower levels of donor cell reconstitution (28.3% reduction at 20 weeks) ( Figure 19E ), and decreased myeloid cell production (24.5% to 17.1%) ( Figure 19F ). In order to further study N-cad + To determine whether niche cells contribute to the long-term self-renewal of HSCs, a secondary transplant was performed 20 weeks after the initial transplant. + BM cells from stromal cell ablated mice had profoundly reduced donor cell reconstitution capacity in secondary transplantation (40.5% reduction at 16 weeks) ( Figure 19G ), although they are capable of multi-lineage reconstruction ( Figure 19H ).

[0315] In addition, we found that N-cad + Conditional knockout of Cxcl12 in stromal cells significantly reduced pHSCs (48% reduction) and ST-HSCs (28.8% reduction), but no significant reduction was observed in rHSCs, although there was a slight, insignificant increase in MPP. Figure 19I ). From N-cad + Conditional deletion of SCF in stromal cells significantly reduced rHSCs (by 60%), pHSCs (by 38.6%), and ST-HSCs (by 53.7%), but slightly increased MPPs ( Figure 19J Overall, we provide the first functional evidence that N-cad + Niche cells promote the maintenance of HSCs, including rHSCs, by producing maintenance factors.

[0316] Example B-4

[0317] Transcriptome analysis of hematopoietic cells and their BM niche cells

[0318] To understand the molecular mechanisms that control how different niche cells contribute to the regulation of HSC subsets, transcriptome profiling was performed on four types of hematopoietic stem and progenitor cells (HSPCs) during steady-state and on rHSCs at day 3 after 5FU, as well as on ten types of BM niche cells. BM niche cells were harvested from different niche regions of the endostium (B) and central bone marrow (M). Figure 26A-B). Pearson distance tree and principal component analysis (PCA) data showed that rHSCs and pHSCs shared unique transcriptome profiles compared with ST-HSCs and MPPs ( Figure 20A Interestingly, 5FU-treated rHSCs appeared to be closer to pHSCs ( Figure 20B ), suggesting that surviving rHSCs are primed for activation to support subsequent hematopoietic regeneration after chemotherapy.

[0319] It has been found that rHSCs are enriched for most of the published HSC-specific markers, such as Slamf1 (CD150), H19, Ctnnal1 (α-Catulin), Fgd5, vWF, Tek (Tie2), Procr (Epcr), Hoxb5, and Meg3 (or Gtl2) (Acar et al., 2015; Chen et al., 2016; Qian et al., 2015; Sanjuan-Pla et al., 2013; Venkatraman et al., 2013). In particular, the vWF in rHSCs was 6.1 times higher than that in pHSCs, indicating that rHSCs are enriched for vWF, which is mostly located at the top of the HSC hierarchy. + HSCs (Sanjuan-Pla et al., 2013). Consistently, progenitor cell signature genes such as CD34, CD48, Flt3 (Flk2), and Itga2 (CD49b) are lowly expressed in rHSCs (Acar et al., 2015; Chen et al., 2016; Gazit et al., 2014). Interestingly, rHSCs after 5FU have high expression levels of CXCR4, Ctnnal1 (α-Catulin) (Park et al., 2002), Esam (endothelial cell selective adhesion molecule), and CD150 (encoding Slamf1, a signaling lymphocyte activation molecule), consistent with their function in the switched primed state ( Figure 20E ).

[0320] In the niche cell analysis, N-cad-driven tdTomato (N-cad-TdT) (M) was found to be the most abundant gene in the Pearson distance tree ( Figure 20C ) and PCA analysis ( Figure 20D ) have very similar transcriptome profiles compared with other niche cells with mesenchymal stem cell (MSC) potential, such as LepR, Cxcl12-RFP, and nestin-GFP cells, indicating that N-cad + The cells may have MSC potential and have similar functions in regulating HSCs. Interestingly, NG2-RFP cells are similar to Col2.3-GFP cells. + Osteoblasts have very similar transcriptome profiles ( Figure 20C-D). NG2-RFP cells were also found to be primarily confined to the endosteum in the epiphysis or diaphysis, but they were also detected in periarterial areas ( Figure 26C ).

[0321] Pecam (CD31) and CDH5 (VE-cadherin) have been found to be enriched in Pecam-GFP + Endothelial cells. PF4 and Adgre1 were enriched in MKs and macrophages (MACs). Cspg4 (NG2) was enriched in NG2-RFP cells. Nestin-GFP cells did not have endogenous nestin (Nes) expression, which is consistent with previous reports (Ding et al., 2012; Greenbaum et al., 2013). Interestingly, several marker genes such as Kitl (SCF), LepR, Cdh2 (N-cadherin, N-CAD), Cxcl12, and Pdgfrα were found to be widely expressed in perivascular niche cells ( Figure 20F Compared with other cells from the endosteal region (e.g., Col2.3-GFP and NG2-RFP cells), N-cad-TdT (B) cells isolated from the endosteal region also expressed high levels of SCF, Cxcl12, LepR, and Pdgfrα ( Figure 20F Our GO term analysis showed that Col2.3-GFP, Pecam-GFP, MK, and Mac cells were highly enriched for RNA metabolism, protein metabolism, other metabolic pathways, and translation activity, indicating that these cells were in a relatively active functional state compared to N-cad-TdT cells and other perivascular area cells that were in a relatively low metabolic state. Endothelial cells and perivascular cells have immune system processes and stress response activities ( Figure 20G ). Due to N-cad + The transcriptome profile of cells is similar to that of other MSCs, such as LepR + , Cxcl12-RFP, and Nestin-GFP cells were similar, so we next analyzed and compared matrix development-related genes from cells from different niches ( Figure 20H). Col2.3-GFP cells were found to be enriched for the osteoblast gene Col1a and the progenitor gene Ly6a (SCA1) (Yang et al., 2014). N-Cad-TdT(B) cells from the endosteal region were enriched for the chondrocyte genes Spock1, Col2a1, and Col1a2, as well as bone development genes (such as Tnc). Interestingly, N-Cad-TdT(B) and N-Cad-TdT(M) were enriched for most mesenchymal stem and progenitor cell (MSPC) genes, such as Prrx1, Pdgfrβ, Pdgfrα, Sp7, Sox9, and Grem1. Bone marrow-harvested NG2-RFP cells also had higher levels of MSPC gene expression, such as Alcam, Sp7, Mcam, Sox9, and Gli1, but had high levels of osteoblast- and chondrocyte-related genes, such as Col1a1, Col2a1, and Col1a2. It was also found that LepR + Cells were uniquely enriched for Gla as well as several osteoblast and chondrocyte markers, such as Atf4 and Tnc. Cxcl12-RFP cells were significantly enriched for the MSPC gene Itgav. Furthermore, Grem1 was enriched in N-Cad-TdT (B) and all perivascular niche cells, but not in NG2-RFP cells.

[0322] Consistent with earlier data, Col2.3-GFP cells were enriched for more mature bone lineage genes (Dmp1, Col1a1, Spp1, Bglap). Nestin-GFP, Cxcl12-RFP, and LepR are consistent with their known roles in enriching MSC genes (Prrx1, Pdgfrα, Itgb1, Grem1) ( Figure 20H Interestingly, in addition to expressing MSC genes, N-Cad-TdT(M) and N-Cad-TdT(B) were enriched in chondrogenic (Sox9, Col2a1 and Tnc), adipogenic (Cebpα, Pparγ, Adipoq) and osteogenic (Col1a1, Runx2) genes, indicating that N-Cad + Tripotential properties of stromal cells. Unexpectedly, NG2-RFP was enriched in bone development genes (Dmp1, Bglap, Sp7, Runx2, Col1a1) and cartilage generation genes (Sox9, Col2a1, and Tnc), indicating that it has an osteochondrogenic role ( Figure 26D These data strongly suggest the heterogeneity of MSPCs in BM.

[0323] Example B-5

[0324] N-cad-CreER T Inducible reporter cells with LepR+ and Cxcl12 + Substantial overlap of stem / stromal cells

[0325] To confirm the transcriptional analysis, we performed in vivo lineage tracing of N-cad using TdT or ZsG reporters and found that N-cad-CreER T Lineage-traced cells partially overlap with Cxcl12-RFP and Nestin-GFP cells ( Figure 20I ). In addition, 98.3% and 97.9% of N-cad-CreER T The derived cells were positive for LepR and Pdgfrα expression ( Figure 20J This result further supports the + Stromal cells have MSC potential as suggested by transcriptome analysis. + To determine the lineage potential of stromal cells, a colony-forming unit-fibroblast (CFU-F) assay was performed to test their proliferation capacity. It was found that most reported niche cells (whether from the endosteal or perivascular zones) had CFU-F activity in less than one in 10 cells, and nestin-GFP was expressed. + The exception is cells, which had CFU-F activity in only one of 17.6 cells. N-cad from bone + One out of 8.79 cells had CFU-F activity. T One in 10.7 bone cells from the bone marrow showed CFU-F activity, and N-cad-CreER T One out of 7.37 bone marrow cells had CFU-F activity. Figure 20K Most CFU-F colonies maintained tdTomato signal ( Figure 21A -B), which indicates that N-cad + The cells are the main source of MSPCs with CFU-F activity.

[0326] Example B-6

[0327] N-cad + Stromal cells generate osteoblasts, chondrocytes, and adipocytes in vitro and in vivo

[0328] To test N-cad + The MSC potential of cells can be determined by + The cells were divided into three aliquots and then subcloned into cultures that allowed bone, fat or chondrocyte differentiation for in vitro differentiation assays.+ The cells undergo multilineage differentiation, giving rise to alkaline phosphate-positive osteoblasts, Oil Red O-positive adipocytes, and aggrecan-stained and toluidine blue-positive chondrocytes ( Figure 21C -E).

[0329] To characterize N-cad + To investigate the in vivo function of stromal cells, we analyzed the dynamic anatomical distribution of N-cad-derived cells after treatment with one dose of TMX. Figure 21F Interestingly, N-cad-derived cells were detected in the posterior metaphysis of trabecular bone as early as 6 h after TMX, but only a few cells were observed in the central bone marrow, suggesting that most N-cad + Cells originate from the endosteal region ( Figure 21H It was also observed that the number of N-cad-derived cells in the trabecular bone region increased by 2.2 ± 0.2-fold 1 to 2 weeks after TMX treatment and remained stable thereafter; in the cortical bone region, the number of cells increased by 2.95 ± 0.2-fold 2 weeks after TMX but then decreased 6 weeks after TMX, indicating that N-cad in the trabecular bone region is more abundant than in the cortical bone region. + Furthermore, N-cad-derived cells continued to increase in the central bone marrow up to 6 weeks, suggesting that N-cad-derived cells may proliferate and differentiate within this region ( Figure 21I ).

[0330] Next, in vivo lineage tracing assays were performed and N-cad + Cells produce Col2.3-GFP in a time-dependent manner + Osteoblasts (Dacic et al., 2001) Figure 22A , Figure 27A -B). As early as 6 hours after TMX induction ( Figure 22B ) in the peritrabecular region and in the compact bone region 14 h after TMX induction ( Figure 22C ) detected N-cad-derived osteoblasts. Further analysis showed that 6 hours after TMX, immature N-cad + Col2.3 – Cells were 10-fold more abundant in the cortical bone region (1.1% ± 0.2%) than in the cortical bone region (0.11% ± 0.04%). Figure 22D , E). Consistently, more immature N-cadherin was enriched in the trabecular bone region compared with the compact bone region 24 h and 2 weeks after TMX induction. + Col2.3 – cell( Figure 22F). 4 weeks after TMX induction, the majority of Col2.3-GFP cells (72% ± 6%) were derived from N-cad + cell( Figure 22G ).

[0331] It was further observed that 4 weeks after TMX injection, N-cad + Stromal cells give rise to adipocytes in the trabecular bone region, particularly endosteal cells ( Figure 22H ), and this was further confirmed by BODIPY lipid probe staining ( Figure 22I ). Interestingly, N-cad + The frequency of adipocytes derived from TMX increased rapidly initially and then decreased, as evidenced by quantitative analysis, in which 39.3% ± 4.5%, 77.1% ± 6.7%, and 17.2% ± 3% of N-cad were observed at 6, 14, and 24 h after TMX induction, respectively. + cells of origin, suggesting that adipocytes may have a frequent turnover rate ( Figure 22J -K, Figure 27C -E). Overall, the data suggest that N-cad + Stromal cells give rise to osteoblastic and adipocyte lineages in vivo.

[0332] Furthermore, Ncad+MSCs were found to be enriched >10-fold compared to other markers. Figure 30 As shown, only 10KNcad+hMSCs produced even higher levels of human thrombopoietin (THPO or TPO) in vivo than total hMSCs. Similarly, Ncad-hMSCs exhibited similar functions to total hMSCs. Therefore, isolating MSCs with N-cadherin antibodies can provide enrichment of the MSC population.

[0333] Example B-7

[0334] N-cad + Stromal cells generate chondrocytes during development and after injury

[0335] Chondrogenesis is active during fetal development and less active in adults (Raghunath et al., 2005; Sophia Fox et al., 2009). T Aggrecan in the femur of R26-tdT mice + Tomato expression was not detected in chondrocytes ( Figure 28A ), although Tomato +Osteoblasts and chondrocytes are adjacent to the growth plate. N-cad-CreER was induced with TMX at 1 or 2 weeks of growth plate development. T ; R26-tdT mice, Tomato in the femoral or tibia cartilage + Chondrocytes were still not detected (data not shown). At embryonic day E12.5, when MSCs undergo condensation and chondrocyte differentiation in fetal bone, N-cad-CreER was first expressed. T ; R26-tdT mice were induced with TMX ( Figure 23A ). It was found that most of the undifferentiated N-cad-derived tdT in E14.5 embryos + Cells are located in the periphery, while differentiated N-cad-derived Tomato + Chondrocytes are located in the central region of the ribs ( Figure 23B Importantly, at P2, N-cad-derived chondrocytes were detected in both the columnar and superficial regions of the femoral trabecular region, where the second ossification center is formed, which is essential for resisting absolute force for deep protection ( Figure 23C , D). Consistently, N-cad-derived adipocytes were detected at 2 months after birth and until 10 months after birth ( Figure 23E , Figure 28B ), indicating that N-cad + Cells contribute to early and long-term adipogenesis during development. However, N-cad was detected at 2 months after birth. + Cell-derived osteopontin + osteoblasts and osteocytes, but not detected in the early P2 stage ( Figure 23F , Figure 28C These data indicate that N-cad-derived embryonic cells give rise to all three osteo-adipogenic-chondrogenic cell lineages and that only embryonic N-cad + In order to effectively form cartilage cells.

[0336] Next, we studied the expression of N-cad in adult mice after injury. + Whether cells can generate chondrocytes depends on whether they are treated with TMX-induced N-cad-CreER at E12.5. T ; Cartilage perforation was performed in R26-tdT mice ( Figure 23G ). 2 to 3 weeks after cartilage injury, we found that N-cad-derived chondrocytes accumulated in the healing tissue at the injury site, increasing 2-fold compared with uninjured control mice ( Figure 23H -I). Due to articular cartilage damage in mice after surgery, the femoral tibial joints swelled ( Figure 28D Alcian blue-positive cells with typical chondrocyte characteristics increased significantly in the post-injury area ( Figure 23J), which indicates that N-cad + MSCs rapidly regenerate chondrocytes in response to injury.

[0337] Taken together, the data demonstrate that N-cad-derived cells induced at the fetal stage (E12.5) can generate chondrocyte progenitors in adults and support chondrocyte regeneration in response to injury.

[0338] discuss

[0339] rHSCs and pHSCs

[0340] The heterogeneity of HSC has been extensively studied. HSC can remain in an active, static or deep static state. It is well known that the static characteristics of HSC are functionally related to their long-term self-renewal potential (Foudi et al., 2008; Wilson et al., 2008). However, how static HSC groups overcome the consequences of bone marrow ablation in vivo remains to be solved. Regardless of their static state, most HSCs cannot survive under chemotherapeutic stress such as 5FU (Longley et al., 2003). According to aspects of this article, it was found that a small part of HSC subpopulations can survive under the 5FU treatment of the first mouse and have resistance to 5FU treatment in a transplant model. Therefore, this HSC subpopulation is defined as rHSC, while other static but chemotherapy-sensitive HSC subpopulations are defined as pHSC (Li and Clevers, 2010). Although these two HSC subpopulations support long-term hematopoiesis in transplant experiments, pHSC rarely produces rHSC, and the latter can produce pHSC. Mechanistically, we found that rHSCs have a weakened DNA repair system compared with pHSCs in steady state, but rHSCs can rapidly activate their DNA repair pathways and stress response programs to survive chemotherapy stress and support subsequent hematopoiesis to overcome the consequences of bone marrow ablation.

[0341] Niche is important in conferring resistance to chemotherapy

[0342] To explore the extrinsic mechanism of chemoresistance of rHSCs, the idea that they are maintained in a specific microenvironment of the BM was tested. By using whole-mount HSC staining (Kunisaki et al., 2013), it was first observed that the majority of HSCs were associated with blood vessels and MKs, as previously reported. However, it was surprisingly found that during steady state and after 5FU treatment, rHSCs were mainly associated with the endosteal niche compared to pHSCs. This suggests that the endosteal niche may form a unique BM microenvironment to protect rHSCs against chemotherapeutic stress. Consistently, upon chemotherapeutic stress, the majority of rHSCs were located in a region rich in chemoresistant N-cad +Stromal cells are protected in the endosteal niche, while blood vessels and perivascular cells are sensitive to 5FU treatment, resulting in a large loss of pHSCs induced by chemotherapy. Whole-mount HSC staining was performed in the sternum, which has abundant bone branches and extensions within the bone marrow. This feature makes it very similar to the trabecular bone region in the femur. Transplantation assays showed that N-cad + This view is supported by the fact that depletion of niche cells affects the maintenance of HSCs, including rHSCs. HSC quiescence is also associated with their hypometabolic state, which is considered similar to "sleep" and has been referred to as HSC dormancy or hibernation (Takubo et al., 2013; Wilson et al., 2008; Yamazaki et al., 2011). However, various aspects of this paper further suggest that quiescence is not the only mechanism that causes drug resistance; rather, intrinsic stress response programs and extrinsic niche protection both contribute to drug resistance.

[0343] Identity and function of N-cad stromal cells in HSC maintenance in the BM niche

[0344] N-cad + Stromal cells were the first HSC niche cells to be identified (Zhang et al., 2003) and subsequently confirmed (Arai et al., 2004; Sugiyama et al., 2006). + The cells were initially proposed to be osteoblast progenitors based on their endosteal location. + Stromal cells were distributed in the endosteal region and perivascular sites. More interestingly, most N-cad + Cells and LepR + Cells and Pdgfrα + Cell overlap. Transcriptional analysis showed that N-cad + cells, LepR + cells, Cxcl12-RFP (CAR) cells, and Nestin-GFP cells have very similar gene expression patterns. Our data strongly suggest that the long-standing debate over the HSC niche concept is likely due to the use of different cell markers rather than their cellular properties.

[0345] To determine N-Cad + The identity of stromal cells was determined by visualizing their regional distribution relative to other known niche cells in the bone marrow using different niche reporter mice. + Stromal cells produced 72% ± 6% of Col2.3-GFP + cells; however, 9.3% ± 4.7% N-cad was also detected in the endosteal region +Col2.3GFP - These immature cells constitute primitive MSCs, which may explain the insufficient efficiency of Col2.3-Cre genetic models in studies of HSC niche function (Ding and Morrison, 2013; Ding et al., 2012; Greenbaum et al., 2013). + and Nestin-GFP + All of them are enriched in trabecular bone, but N-Cad-TdT + The HSCs were localized in the trabecular bone region of the implantation where they were detected, as previously reported (Nilsson et al., 2001; Xie et al., 2009), and survived after stress, as observed here. All these data suggest that, despite the expression of N-cad + The cells have similar transcriptomic profiles to other MSCs, but their anatomical distribution may indicate that they have unique HSC niche functions; indeed, it has been shown that N-cad + Endosteal niche cells play a key role in the preservation of rHSCs.

[0346] By using the inducible DTR system, N-cad was discovered + Ablation of these cells eliminated pHSCs and rHSCs from the BM. This was achieved through N-cadherin in the endosteal and perivascular regions. + The anatomical distribution of niche cells can be explained. In addition, it has been shown that N-cad expression can be detected in a subset of HSCs; however, the N-cad-TdT reporter mouse line does not support this observation (data not shown). This can be partly explained by the discordance between their protein and transcript levels, as another N-cad-mCherry mouse line (a fusion at the protein level) does have a small HSC subset (CD49b - CD34 - Flt2 - LSK) showed low levels of N-cad expression (preliminary observation). Functional transplantation data showed that N-cad + Ablation of niche cells leads to a decrease in HSCs (including rHSCs). + In cells lacking Cxcl12 and SCF, N-cad + Stromal cells promote the maintenance and regulation of HSCs by producing these two factors. Overall, the aspects of this paper demonstrate that N-cad + Stromal cells serve as MSCs and support primitive HSC maintenance, especially under stress.

[0347] Transplantation of hematopoietic stem cells (HSCs) from human umbilical cord blood (hUCB) holds great promise for treating various blood diseases, including cancer, but the limited number of HSCs in a single hUCB unit limits its widespread use. Although extensive efforts have been made to develop various methods for ex vivo expansion of human HSCs by targeting single molecules or pathways, it is unknown whether simultaneous manipulation of a large number of targets essential for stem cell self-renewal can be achieved. Recent studies have shown that N 6 -methyladenosine (m 6 A) Regulates the expression of a group of mRNAs that are crucial for stem cell fate determination by affecting their stability. 6 A. It is well known that Ythdf2 promotes targeted mRNA decay. However, the physiological function of Ythdf2 on adult stem cells remains unclear. The embodiments herein demonstrate that conditional knockout (KO) of Ythdf2 in mice increases the phenotype and number of functional HSCs, but neither reverses lineage differentiation nor leads to hematopoietic malignancies. In addition, knockdown (KD) of human YTHDF2 results in a greater than 10-fold increase in the ex vivo expansion of hUCB HSCs, a 5-fold increase in colony-forming units (CFUs), and a greater than 8-fold increas...

Claims

1. An in vitro method for expanding a stem cell population obtained from a tissue selected from peripheral blood, umbilical cord blood and bone marrow, the method comprising regulating the N 6 -methyladenosine (m 6 A) mRNA modification pathways to expand stem cell populations, The stem cells are selected from hematopoietic stem cells (HSC), mesenchymal stem cells (MSC) and a combination thereof. Among them, adjust m 6 A mRNA modification pathway includes knocking out or knocking down the gene expressing Ythdf2.

2. The method of claim 1, wherein adjusting m 6 A mRNA modification pathway includes exposing the stem cell to an Mx1-Cre targeting system, which inactivates or deletes at least a portion of the gene expressing Ythdf2.

3. The method of claim 1, wherein adjusting m 6 A mRNA modification approach includes introducing shRNA into stem cells to reduce the expression of the Ythdf2-expressing gene mutation.

4. The method of claim 3, wherein the shRNA is introduced by exposing the stem cells to a lentivirus to deliver the shRNA.

5. The method of claim 1, wherein the knockout of the gene expressing Ythdf2 is generated using Crispr-Cas9 technology. The method of claim 1 , wherein the stem cells are expanded at least two-fold.

7. The method of claim 6, wherein the stem cells are expanded by at least 4-fold.

8. The method of claim 7, wherein the stem cells are expanded at least 5-fold.

9. The method of claim 8, wherein the stem cells are expanded at least 8-fold.

10. The method of claim 9, wherein the stem cells are expanded by at least 10-fold.

11. The method of claim 1, wherein the stem cell is an HSC.

12. The method of claim 1, wherein the expanded stem cells have at least a 5-fold increase in total colony forming units (CFU).

13. The method of claim 1, wherein the expanded stem cells have at least a 3.8-fold increase in CFU-granulocyte erythrocyte monocyte megakaryocyte (GEMM) colonies.

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