Mesenchymal stem cell as well as culture method and application thereof

Abstract

The invention relates to the technical field of cell culture, in particular to mesenchymal stem cells as well as a culture method and application thereof. Specifically, the invention relates to a mesenchymal stem cell derived from brain organs, and the mesenchymal stem cell also highly expresses SFRP1 and SFRP2 in addition to a mesenchymal stem cell marker. The present invention also relates to a method for producing mesenchymal stem cells derived from brain organs from human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC), which obtains human mesenchymal stem cells from brain organs produced from human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) using an improved brain organ culture method. The invention also relates to the application of the mesenchymal stem cell in supporting the amplification of the hematopoietic stem cell, maintaining the transportability of the hematopoietic stem cell and improving the microenvironment maintained by the hematopoietic stem cell.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a mesenchymal stem cell and a culture method and application thereof. Background Art

[0002] Human hematopoietic stem cells can be easily obtained from umbilical cord blood, but the number of hematopoietic stem cells from umbilical cord blood is small and their use is limited by the age and weight of the recipient. Although in vitro expansion of hematopoietic stem cells is necessary, this goal has been difficult to achieve.

[0003] Mesenchymal stem cells from bone marrow or umbilical cord are usually used as feeder cells, which can maintain the stemness of hematopoietic stem cells in vitro, but have limited ability to expand hematopoietic stem cells in vitro. Since bone marrow or umbilical cord mesenchymal cells cannot proliferate indefinitely in vitro and have individual differences, they cannot form stable hematopoietic stem cell expansion cells.

[0004] Hematopoietic stem cells (HSCs) exist in a heterogeneous microenvironment (or niche) where they give rise to all functional mature blood cells. HSCs are present in different tissues during individual development, with adult hematopoiesis first appearing in the yolk sac, followed by the AGM region. The fetal liver is the site of expansion of adult HSCs, and then after birth, they colonize the bone marrow. In parallel with the AGM region during the fetal period, HSCs have also been found in the head of mouse embryos (Li, Lan et al., 2012). The HSC niche consists of several different cell types, including vascular cells, mesenchymal stem cells / stromal cells (MSCs), and neural cells, which strictly regulate the self-renewal capacity and multipotency of HSCs (Morrison & Spradling, 2008). It is generally believed that mesenchymal stem cells are a key component of the HSC niche (Crane, Jeffery et al., 2017, Mendelson & Frenette, 2014, Pinho & Frenette, 2019). Adult mouse bone marrow and embryonic mesenchymal cell lines with a mesenchymal phenotype have been cloned and characterized for their ability to support the growth, maintenance, and differentiation of mouse and human hematopoietic stem cells (Moore, Ema et al., 1997; Oostendorp, Harvey et al., 2002; Oostendorp, Robin et al., 2005; Xu, Tsuji et al., 1998). Adult human mesenchymal stem cells, such as brain-derived mesenchymal stem cells (BM-MSCs), placenta-derived mesenchymal stem cells (PAM-MSCs), and umbilical cord-derived mesenchymal stem cells (UC-MSCs), have the ability to serve as donors to maintain the undifferentiated state of hematopoietic stem / progenitor cells (HSPCs), although their efficacy varies depending on the cell source (Crippa & Bernardo, 2018, Mayani et al., 2017, Kadekar, Kale et al., 2015, Lu, Liu et al., 2006). Clinical studies have confirmed that co-transplantation of umbilical cord blood hematopoietic stem cells (UCB-HSPC) with BM-MSC or UC-MSC can enhance hematopoietic cell engraftment (Wu, Cao et al., 2014, Zhang, Chai et al., 2006). However, there are also studies showing that mesenchymal stem cells from different tissues show diversity in a range of biological properties and substantial heterogeneity (Hass, Kasper et al., 2011, Heo, Choi et al., 2016). In addition, due to limited long-term proliferation capacity, the number of mesenchymal stem cells obtained from a single donor source is limited. Therefore, the production of a homogeneous mesenchymal stem cell population with specific therapeutic application standards remains a major challenge.

[0005] Significant efforts have been made to identify alternative cell sources for MSCs, particularly through human pluripotent stem cell (hPSC) approaches. Many recent reports have shown that unlimited production of mesenchymal stem cells can be achieved using HSC-derived mesodermal cells, neural crest cells (NCCs), and trophoblast-like intermediate cells (Chin, Cooper et al., 2016; Kamiya, Takenaka-Ninagawa et al., 2022; Vodyanik, Yu et al., 2010; Wang, Lazorchak et al., 2016). These hPSC-derived MSCs (hPSC-MSCs) represent a possible alternative source of therapeutic MSCs that can overcome the problems associated with standardized cell preparation and ready-made production in clinical applications. Mesodermal or unknown-origin hPSC-MSCs provide a supportive feeding layer for the self-renewal and colony-forming capacity of CD34+HSPCs in vitro and in vivo ((Chin, Li et al., 2018, Li, Tormin et al., 2013, Moslem, Eberle et al., 2015).

[0006] Although a variety of methods have been applied to generate mesenchymal stem cells from human hematopoietic stem cells, most of them have been shown to be inefficient. Typically, these methods rely on purifying MSCs by flow cytometry sorting (FACS) using specific cell surface markers (Islam et al., 2015), culture media containing fetal bovine serum (Wang et al., 2016, Zhao, Gregory et al., 2015), and monolayer differentiation culture systems (Lian, Zhang et al., 2010), all of which can lead to batch-to-batch variability, functional heterogeneity, and inefficiency.

[0007] Mesenchymal stem cells (DP-MSCs) derived from dental pulp tissue differ from other mesenchymal stem cells because their embryonic origin is the neural crest (Stanko, Altanerova et al., 2018). Mouse DP-MSCs have been reported to support the in vitro growth of human UCB-HSPCs (Nakatsuka, Matsuoka et al., 2015). Despite the importance of NCCs in developmental biology and disease, the isolation of NCCs of human origin remains elusive. Successful isolation, propagation, and directed differentiation of NCCs from human hematopoietic stem cells by conventional monolayer culture have been reported (Lee, Kim et al., 2007, Li, Huang et al., 2018). However, the development of fully mature functional cells from human hematopoietic stem cells is a challenge. The recently developed 3D organoid approach may help promote further diffusion of nutrients and differentiation factors through the scaffold to the embedded cells, thereby helping the development of fully functional mature cells (Lin, Homan et al., 2019). There are many reports that hPSC-derived organoids can be used to construct a variety of simulated human tissues and organs, including liver, bone, intestine, kidney, retina, and brain (from & Spence, 2021). Recently, the generation of functional NNCs has been reported, and their properties have been well characterized in chicken, mouse, and human using 3D methods (Bilozur & Hay, 1988, Han, King et al., 2022, Namkoong, Güven et al., 2016). 3D floating sphere culture also maintains the neural crest-related properties of human dental pulp stem cells (Pisciotta, Bertoni et al., 2018), and NC-derived stem cells generated using 3D culture show genetic stability and maintain stemness characteristics (Greiner, Hauser et al., 2011). Summary of the invention

[0008] To solve the above problems, the inventors studied a new method for obtaining stromal cells from hPSC (including hESC and hiPSC). These long-term cultured organoid-derived stromal cells have differentiation characteristics of neural crest cell lineage. They express high levels of MSC-specific markers and exhibit neural cell characteristics, so we named them brain organoid-derived mesenchymal stem cells (pMSC). These pMSCs not only mimic the morphology and function of UC-MSCs, but also express additional functional molecules involved in regulating HSPC expansion and interaction. When UCB-CD34+HSPCs were co-cultured with UC-MSCs or pMSCs, the latter led to strong expansion of human UCB-CD34+HSPCs in long-term culture and effectively maintained their transplantability. RNA sequencing results showed that co-culture of human UCB-CD34+HSPCs with pMSCs can improve the microenvironment for HSC maintenance. pMSCs highly express Wnt signal inhibitors SFRP1 and SFRP2, indicating that they may help regulate the cell cycle and promote the expansion / maintenance of CB-CD34+HSPCs by antagonizing Wnt activation. Unlike conventional 2D culture methods, our method allows all multi-step differentiation processes to be completed in a 3D vessel without the need for cell passaging. pMSCs generated by this method could serve as a good cell source for future clinical use as a standardized treatment for hematopoietic disorders.

[0009] Specifically, in one aspect, the present invention provides a brain organoid-derived mesenchymal stem cell having the following characteristics: (i) >94% of the cells express CD73, CD105, CD44, CD29, and CD90; (ii) <1% of the cells express CD11b, CD34, CD45, and CD144; (iii) compared with mesenchymal stem cells from other sources, they express higher levels of SFRP1.

[0010] Furthermore, <1% of the mesenchymal stem cells express CD271.

[0011] Furthermore, >99.8% of the mesenchymal stem cells express CD73, CD105, CD44 and CD29.

[0012] Furthermore, >94.2% of the mesenchymal stem cells express CD90.

[0013] Furthermore, the mesenchymal stem cells from other sources may include, but are not limited to, brain-derived mesenchymal stem cells (BM-MSC), placenta-derived mesenchymal stem cells (PAM-MSC) and umbilical cord-derived mesenchymal stem cells (UC-MSC).

[0014] As used herein, SFRP1 refers to secreted frizzled-related protein 1 (SFRP1), which is a Wnt signaling pathway negative regulator well known in the art. In addition to SFRP1, the mesenchymal stem cells described herein also express higher levels of other Wnt signaling pathway negative regulators compared to mesenchymal stem cells from other sources.

[0015] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express a higher level of SFRP2, a negative regulator of the Wnt signaling pathway.

[0016] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express a higher level of IGFBP2, a negative regulator of the Wnt signaling pathway.

[0017] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of the negative regulators of the Wnt signaling pathway, DKKL1, FRZB, SOX9, DACT3, SOX17, DKK3 and FRMD8.

[0018] In addition, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells described herein also express higher levels of one or more other characteristic functional molecules.

[0019] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of at least one of CD24 and IL17RD.

[0020] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of COLEC12, FOXC1 and TGA8.

[0021] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of NPTX1, NES, CXCL12, NFATC4, SIX1, CLSTN2, SCG2, MSX2, MDFI, TFAP2A, APOE, BMF, CPXM1, NYNRIN, DSP, CPE, PCDHB5, SNED1, AUTS2, DLX1, SESN3, AFF3, PALM, SENP7, FREM2, PCDHB2, ADAMTS10, TFAP2B, KIAA1755, DLX2, IGSF3, SHC2, TSPAN11, PLXNA4, TMEM246, VANGL2 and FAM131B.

[0022] In addition, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells described herein also express higher levels of factors that regulate the maintenance of HSC. For example, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of MCAM, NES, KITLG, JAG1, IGFBP2 and TNC.

[0023] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of CCDC126, SCG2, PSAP and COL18A1.

[0024] Furthermore, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells express lower levels of genes involved in regulating stem cell differentiation, IL-6 production, cytokine production, and neutrophil migration.

[0025] In another aspect, the present invention provides a co-culture of brain organoid-derived mesenchymal stem cells and hematopoietic stem cells as described herein.

[0026] Furthermore, the mesenchymal stem cells support the expansion of the hematopoietic stem cells, maintain the transplantability of the hematopoietic stem cells, and improve the microenvironment maintained by the hematopoietic stem cells.

[0027] Furthermore, the hematopoietic stem cells include hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem progenitor cells derived from umbilical cord blood and hematopoietic stem cells derived from bone marrow.

[0028] Furthermore, compared with the co-culture of mesenchymal stem cells and hematopoietic stem cells from other sources, the expression levels of CD38, CSF1R and ELANE progenitor cell characteristic genes in the hematopoietic stem cells are reduced.

[0029] Furthermore, compared with the co-culture of mesenchymal stem cells and hematopoietic stem cells from other sources, the expression levels of HOXA9, HLF and KIT HSC characteristic genes in the hematopoietic stem cells are increased.

[0030] Furthermore, the co-culture has more CD34+CD38-CD90+CD45RA- long-term HSCs, CD34+CD38-CD90-CD45RA- short-term HSCs and CD34+CD38-CD90-CD45RA+ committed progenitor cells compared with the co-culture of mesenchymal stem cells and hematopoietic stem cells from other sources.

[0031] In another aspect, the present invention provides a method for generating brain organoid-derived mesenchymal stem cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs), the method comprising the following steps:

[0032] (1) hESCs or iPSCs are suspended in Stage 0 medium containing Y-27632, and then cultured in Stage 1 neural induction medium, Stage 2 neural differentiation medium, and Stage 3 neural differentiation medium to generate brain organoids.

[0033] (2) deriving mesenchymal stem cells from the brain organoids,

[0034] Wherein step (2) comprises:

[0035] In the process of culturing brain organoids, adherent cells derived from brain organoids are cultured and expanded in MSC culture medium to obtain mesenchymal stem cells, or

[0036] The brain organoid is dispersed into single cells, and the single cells of the brain organoid are cultured in an MSC culture medium, thereby obtaining mesenchymal stem cells.

[0037] Further, in step (1), hESCs or iPSCs are cultured in Stage 1 neural induction medium on days 2-5, in Stage 2 neural differentiation medium on days 6-24, and in Stage 3 neural differentiation medium on days 25-43.

[0038] Furthermore, the Stage 0 culture medium is a ncTarget culture medium containing Y-27632.

[0039] Furthermore, the Stage 1 neural induction medium is a DMEM / F12 medium containing 10-20% (vol / vol) KSR, 1-5 μM Dorsomorphin and 5-10 μM SB-431542.

[0040] Furthermore, the Stage 2 neural induction medium is Neurobasal A medium containing 10-20 ng / ml EGF and 10-20 ng / ml FGF2.

[0041] Furthermore, the Stage 3 neural induction medium is Neurobasal A medium containing 10-20 ng / ml BDNF and 10-20 ng / ml NT3.

[0042] Furthermore, the Stage 1, Stage 2 and Stage 3 culture media may also contain other components, including but not limited to non-essential amino acids, B27 additives, GlutaMAX additives, 2-mercaptoethanol, antibiotics, etc.

[0043] Furthermore, in step (2), the adherent cells are generated from day 21 to day 39 of the culture process.

[0044] Further, in step (2), on day 43, the generated brain organoids are dispersed into single cells.

[0045] Furthermore, in step (2), the brain organoids can be dispersed into single cells by any conventional method known in the art, for example, the brain organoids can be generated by trypsin digestion.

[0046] Furthermore, the MSC culture medium may be a conventional culture medium for maintaining MSCs well known in the art. For example, an example of such a MSC culture medium may be nsMissionhMSC culture medium V3.0 or NCmisstion culture medium purchased from Zhongsheng Suyuan.

[0047] Furthermore, the brain organoid-derived mesenchymal stem cells produced by the method described in this article have the following characteristics: (i) >94% of the cells express CD73, CD105, CD44, CD29, and CD90; (ii) <1% of the cells express CD11b, CD34, CD45, and CD144; (iii) compared with mesenchymal stem cells from other sources, they express higher levels of SFRP1.

[0048] Further, <1% of the brain organoid-derived mesenchymal stem cells generated by the methods described herein expressed CD271.

[0049] Furthermore, >99.8% of the brain organoid-derived mesenchymal stem cells produced by the methods described herein expressed CD73, CD105, CD44, and CD29.

[0050] Furthermore, >94.2% of the brain organoid-derived mesenchymal stem cells generated by the method described herein expressed CD90.

[0051] Furthermore, the mesenchymal stem cells from other sources may include, but are not limited to, brain-derived mesenchymal stem cells (BM-MSC), placenta-derived mesenchymal stem cells (PAM-MSC) and umbilical cord-derived mesenchymal stem cells (UC-MSC).

[0052] Furthermore, compared with mesenchymal stem cells from other sources, brain organoid-derived mesenchymal stem cells generated by the methods described herein also express higher levels of SFRP2.

[0053] Furthermore, compared with mesenchymal stem cells from other sources, brain organoid-derived mesenchymal stem cells generated by the methods described herein also express higher levels of IGFBP2.

[0054] Furthermore, compared with mesenchymal stem cells from other sources, the brain organoid-derived mesenchymal stem cells produced by the methods described herein also express higher levels of one or more of DKKL1, FRZB, SOX9, DACT3, SOX17, DKK3, and FRMD8.

[0055] Furthermore, compared with mesenchymal stem cells from other sources, the brain organoid-derived mesenchymal stem cells produced by the methods described herein also express higher levels of at least one of CD24 and IL17RD.

[0056] Further, compared with mesenchymal stem cells from other sources, the brain organoid-derived mesenchymal stem cells produced by the methods described herein also express higher levels of one or more of COLEC12, FOXC1 and TGA8.

[0057] Furthermore, compared with mesenchymal stem cells from other sources, the brain organoid-derived mesenchymal stem cells produced by the method described in this article also express higher levels of NPTX1, NES, CXCL12, NFATC4, SIX1, CLSTN2, SCG2, MSX2, MDFI, TFAP2A, APOE, BMF, CPXM1, NYNRIN, DSP, CPE, PCDHB5, SNED1, AUTS2, DLX1, SESN3, AFF3, PALM, SENP7, FREM2, PCDHB2, ADAMTS10, TFAP2B, KIAA1755, DLX2, IGSF3, SHC2, TSPAN11, PLXNA4, TMEM246, VANGL2 and FAM131B.

[0058] Further, compared with mesenchymal stem cells from other sources, the brain organoid-derived mesenchymal stem cells produced by the methods described herein also express higher levels of one or more of MCAM, NES, KITLG, JAG1, IGFBP2 and TNC.

[0059] Further, compared with mesenchymal stem cells from other sources, the brain organoid-derived mesenchymal stem cells produced by the methods described herein also express higher levels of one or more of CCDC126, SCG2, PSAP and COL18A1.

[0060] Further, compared with mesenchymal stem cells from other sources, brain organoid-derived mesenchymal stem cells produced by the methods described herein express lower levels of genes involved in regulating stem cell differentiation, IL-6 production, cytokine production, and neutrophil migration.

[0061] Furthermore, brain organoid-derived mesenchymal stem cells generated by the methods described herein support the expansion of hematopoietic stem cells, maintain the transplantability of hematopoietic stem cells, and improve the microenvironment for hematopoietic stem cell maintenance.

[0062] In another aspect, the present invention provides the use of brain organoid-derived mesenchymal stem cells as described herein for supporting the expansion of hematopoietic stem cells, maintaining the transplantability of hematopoietic stem cells, and improving the microenvironment for hematopoietic stem cell maintenance. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 A protocol roadmap for the generation of human cortical organoids from neurally induced hiPSCs is shown.

[0064] Figure 2 Microscopic observation of DAY0 3D spheroid morphology of human cortical organoids generated from neurally induced hiPSCs. Spheroid size ≈ 290 μm.

[0065] Figure 3 The results of microscopic observation of cell morphology during Stage 1 induction of neural-induced hiPSCs to generate human cortical organoids after hCO culture until DAY5 are shown (4X).

[0066] Figure 4 Shown are microscopic observations of cell morphology during Stage 2 induction of neural-induced hiPSCs to generate human cortical organoids cultured in hCO until DAY8 (10X).

[0067] Figure 5 The image shows the microscopic observation results (10X) of cell morphology of human cortical organoids generated from neural-induced hiPSCs cultured in hCO until DAY17.

[0068] Figure 6 The results of microscopic observation of cell morphology during Stage 3 induction of neural-induced hiPSCs to generate human cortical organoids cultured in hCO until DAY22 are shown (4X).

[0069] Figure 7 The results of microscopic observation of cell morphology during Stage 3 induction of neural-induced hiPSCs to generate human cortical organoids cultured in hCO until DAY35 are shown (4X).

[0070] Figure 8 Microscopic observation results (4X) of cell morphology of human cortical organoids generated from neural-induced hiPSCs cultured in hCO until DAY40 and DAY43 during Stage 3 induction.

[0071] Fig. 9The flow cytometry analysis of a pMSC obtained in Example 1 is shown.

[0072] Fig.10 The flow cytometry analysis of a pMSC obtained in Example 1 is shown.

[0073] Fig.11 It was shown that the pMSCs obtained in Example 1 have the ability to support the expansion of hematopoietic stem cells.

[0074] Fig.12 Schematic diagrams and representative micrographs showing typical cell morphology of H1-pMSCs, hiPSC-pMSCs, and UC-MSCs are shown; scale bar, 200 μm.

[0075] Fig.13 The multi-lineage differentiation of pMSCs was shown. After 2 weeks of induction, the differentiated pMSCs were stained with Alizarin Red for mineralization, Toluidine Blue for chondrocytes, or Oil Red for lipid droplets.

[0076] Fig.14 The percentages of surface markers in H1-pMSCs, hiPSC-pMSCs, and UC-MSCs are shown.

[0077] Fig.15 FACS analysis of surface markers in H1-pMSCs, hiPSC-pMSCs, and UC-MSCs is shown.

[0078] Fig.16 Growth curves of UC-MSCs and pMSCs are shown (n=3).

[0079] Fig.17 Center A shows a histogram showing the viability of H1-pMSCs, hiPSC-pMSCs, and UC-MSCs (7-AAD-Annex V-).

[0080] Fig.17 Center B shows the principal component analysis of H1-hESC, hiPSC, H1-pMSC, hiPSC-pMSC and UC-MSC.

[0081] Fig.18 Heat maps showing genes associated with pluripotency and MSCs that are differentially expressed in H1-hESCs, hiPSCs, H1-pMSCs, hiPSC-pMSCs, and UC-MSCs are shown.

[0082] Fig.19 Heat maps showing differentially expressed adipogenic and osteochondrogenic progenitor genes in H1-hESCs, hiPSCs, H1-pMSCs, iPSC-pMSCs, and UC-MSCs are shown.

[0083] Fig. 20 A shows a Venn diagram and GO term analysis showing the number of common and unique up-regulated genes in H1-pMSCs and hiPSC-pMSCs compared with UC-MSCs.

[0084] Fig. 20 Center B shows a heat map showing genes associated with axon development and neural crest cell differentiation that are differentially expressed in H1-pMSCs, hiPSC-pMSCs, and UC-MSCs.

[0085] Fig.21 A heat map showing that genes associated with mesenchymal development were differentially expressed in H1-pMSCs, iPSC-pMSCs, and UC-MSCs is shown.

[0086] Fig. 22 Venn diagrams and GO term analysis showing the number of common and unique up-regulated genes in H1-pMSCs and hiPSC-pMSCs compared with UC-MSCs are shown.

[0087] Fig.23 A shows the experimental design for the isolation of fresh CD34+ HSPCs from human umbilical cord mononuclear cells using CD34 magnetic beads.

[0088] Fig.23 Panel B shows a representative FACS analysis of CD34+ cells in human umbilical cord mononuclear cells sorted by CD34 magnetic beads.

[0089] Fig.24 Panel A shows representative FACS analysis of UCB-CD34+ cells co-cultured with SCF, TPO, and FLT-3L for 7 days with and without H1-pMSCs, iPSC-pMSCs, and UC-MSCs in 5% serum or SFEM medium without added cytokines.

[0090] Fig.24 Panel B shows the yield of CD34+Lin- cells recovered from 7 days of co-culture in 5% serum without added cytokines or in SFEM medium with SCF, TPO, and FLT-3L.

[0091] Fig.25 The total number of cells on days 7-35 after co-culture with and without H1-pMSCs, hiPSC-pMSCs, and UC-MSCs are shown.

[0092] Fig.26 The cell yields of CD34+CD38+Lin-, CD34+CD38-Lin- on days 7-35 after co-culture with and without H1-pMSC, hiPSC-pMSC, and UC-MSC are shown

[0093] Fig. 27 The percentages of CD34+CD38+Lin- and CD34+CD38-Lin- cells from day 7 to day 35 after co-culture with and without H1-pMSCs, hiPSC-pMSCs, or UC-MSCs are shown.

[0094] Fig.28 Representative FACS analysis of CD34+CD38-CD90+CD45RA-LT-HSCs, CD34+CD38-CD90-CD45RA-ST-HSCs, CD34+CD38-CD90-CD45RA+ committed progenitors (C-progenitors), and CD34+CD38+ progenitors on day 7 after co-culture with and without H1-pMSCs, hiPSC-pMSCs, or UC-MSCs is shown.

[0095] Fig.29 The cell yields of CD34+CD38-CD90+CD45RA-LT-HSCs, CD34+CD38-CD90-CD45RA-ST-HSCs, CD34+CD38-CD90-CD45RA+ committed progenitors (C-progenitors), and CD34+CD38+ progenitors on day 7 after co-culture with and without H1-pMSCiPSC-pMSCs or UC-MSCs are shown.

[0096] Fig.30 The results of CFC assay of UCBCD34+HSCs after 7 or 35 days of co-culture with and without H1-pMSCs, hiPS-pMSCs, and UC-MSCs are shown in Figures A and B. The morphology of hematopoietic colonies after 14 days of culture in methylcellulose-based semisolid medium at 37°C and 5% CO2.

[0097] Fig.31 Shown is the quantification of hematopoietic cell apoptosis in four culture conditions by Annexin V / PI staining.

[0098] Fig.32 The experimental design is shown: human umbilical cord mononuclear cells were co-cultured with or without pMSCs and UC-MSCs for 2 weeks and then transplanted into sublethally irradiated BNDG mice. Flow cytometric analysis was used to detect chimerism of human blood cells in the peripheral blood or bone marrow of recipient mice 3 or 6 months after transplantation.

[0099] Fig.33 Center A shows representative flow graphs of human cell (hCD45+) engraftment in PBMCs of BNDG mice 3 months after transplantation with human monocytes co-cultured with or without UCB-MSCs or pMSCs.

[0100] Fig.33Panel B shows the engraftment status (percentage of human CD45+ cells) of mice 3 months after transplantation.

[0101] Fig.34 Representative flow graphs showing human cell (hCD45+) engraftment in PBMCs of BNDG mice 6 months after transplantation with human monocytes co-cultured with or without UCB-MSCs and pMSCs

[0102] Fig.35 Shown are the engraftment (percentage of human CD45+ cells) of mice 6 months after transplantation.

[0103] Fig.36 Multilineage differentiation (CD33+ myeloid cells and CD3+ T cells) of mice 6 months after transplantation is shown.

[0104] Fig.37 Heat maps showing ECM-related genes that are differentially expressed in H1-hESCs, hiPSCs, H1-pMSCs, hiPS-pMSCs, and UC-MSCs are shown.

[0105] Fig.38 Venn diagrams and GO term analysis showing the number of common and unique up-regulated genes in H1-pMSCs and hiPSC-pMSCs compared with UC-MSCs are shown.

[0106] Fig.39 A heat map showing that genes associated with HSC maintenance are differentially expressed in H1-pMSCs, hiPS-pMSCs, and UC-MSCs is shown.

[0107] Fig.40 A shows a Venn diagram, which shows that a total of 1047 genes were down-regulated in H1-pMSCs or hiPSC-pMSCs compared with UC-MSCs.

[0108] Fig.40 Figure B shows GO term analysis, which shows that graphene oxide terms enriched in down-regulated genes in pMSCs compared with UC-MSCs include regulation of stem cell differentiation, IL-6 production, cytokine production, and neutrophil migration.

[0109] Fig.40 C shows principal component analysis (PCA) and Pearson distance tree data, which show that CD34+ cells co-cultured with H1-pMSCs or hiPSC-pMSCs have similar transcriptome profiles to CD34+ cells co-cultured with UC-MSCs or without stromal cells.

[0110] Fig.41GO term analysis of common and unique upregulated genes in CD34+ cells co-cultured with H1-pMSCs, hiPSC-pMSCs, and UC-MSCs compared to cells without stromal cells is shown.

[0111] Fig.42 GO term analysis of common and unique upregulated genes in CD34+ cells co-cultured with H1-pMSCs, hiPSC-pMSCs, and UC-MSCs compared to cells without stromal cells is shown.

[0112] Fig.43 The expression of HSC, HPC, GMP, and MEP signature genes in CD34+ cells co-cultured with and without H1-pMSCs, hiPSC-pMSCs, and UC-MSCs is shown.

[0113] Fig.44 Multiple sample sequencing results of pMSCs are shown.

[0114] Fig.45 Center A shows a heat map showing that genes associated with negative regulation of the Wnt signaling pathway are differentially expressed in H1-pMSCs, hiPSC-pMSCs, and UC-MSCs.

[0115] Fig.45 Center B shows the FPKM of SFRP1, SFRP2, and IGFBP2 expressed in H1-pMSCs, hiPSC-pMSCs, and UC-MSCs.

[0116] Fig.46 Venn diagrams and GO term analysis showing the number of common and unique up-regulated proteins in the culture supernatants of H1-pMSCs and hiPSC-pMSCs compared with UC-MSCs are shown.

[0117] Fig.47 A shows a heat map showing the coordinate regulation of genes in H1-pMSCs and hiPSC-pMSCs in RNA-seq and proteomic analysis.

[0118] Fig.47 Center B shows a volcano plot demonstrating upregulation of SFRP1 and SFRP2 in CD34+ cells co-cultured with H1-pMSCs or hiPSC-pMSCs compared with UC-MSCs.

[0119] Fig.48 The experimental design is shown: UCB-CD34+ cells were co-cultured with pMSCs and UC-MSCs in Transwells by adding 3 μM CHIR99021 or DMSO.

[0120] Fig.49Center AC shows the number of CD34+Lin-, CD34+CD38-Lin-, and Lin+ cells after the addition of 3 μM CHIR99021 and DMSO to activate Wnt signaling.

[0121] Fig.50 Shown are representative FACS analyses of CD34+Lin- cells at day 7 after co-culture with H1-pMSCs, hiPSC-pMSCs, and UC-MSCs in the presence of 3 μM CHIR99021 or DMSO.

[0122] Fig.51 Shown are the percentages of live cells, early apoptotic (E-apoptosis), late apoptotic (L-apoptosis), and dead cells in CD34+Lin- cells at day 7 after co-culture with and without H1-pMSCs, hiPSC-pMSCs, or UC-MSCs after activation of Wnt signaling by the addition of 3 μM CHIR99021.

[0123] Fig.52 Shown are representative flow cytometric analysis and quantification of BrdU incorporation and 7-AAD staining to determine the cell cycle distribution of 34+Lin-HSPCs at day 7 of Transwell culture after addition of 3 μM CHIR99021 to activate Wnt signaling. DETAILED DESCRIPTION

[0124] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0125] Example 1: Generation of pMSCs

[0126] Animal-free and well-defined human brain organoid-derived stromal cell culture method to support hematopoietic stem cell expansion:

[0127] This study intends to induce 3D aggregates of hiPSCs through neural induction and culture in the presence of various small molecules to obtain specific cell fates, so that hiPSCs can produce human cortical organoids (hCO) similar to the dorsal forebrain, also known as the cerebral cortex (hCS). hCOs are then cultured for a certain number of days, digested into single cells, and induced with mesenchymal stem cell (MSC) culture medium to obtain hCO-derived (MSCs). The research plan roadmap is as follows Figure 1 shown.

[0128] The specific methods and steps are as follows:

[0129] 1. Culture hiPSCs until they are 80%-85% confluent, aspirate the culture medium in the culture dish with a Pasteur pipette, and wash once with 2 mL of saline.

[0130] 2. Add Accutase solution (100 μL / cm 2 ) and incubate at room temperature for 5-10 minutes. Make sure the Accutase solution is at the appropriate temperature (15 to 25°C).

[0131] 3. When the cells are completely detached from the culture dish, add double volume of saline (200 μL / cm 2 )Dilute Accutase.

[0132] 4. Transfer the cell solution from the culture dish to a 15 mL centrifuge tube, count the cells, and then centrifuge the cell solution at 400 × g for 5 min.

[0133] 5. Discard the supernatant and resuspend the cell pellet in stage 0 medium (ncTarget+Y-27632).

[0134] 6. Add the cells to a low-adhesion 96-well plate at a certain density, with the cell density per well being 8,000 cells / 100 μL.

[0135] 7. After sedimentation for at least 30 minutes, place the 96-well plate on a 3D shaker and adjust the speed to 60 r / min.

[0136] Among them, the culture medium scheme for each stage:

[0137] Stage 1 Neural Induction Medium (Day 2-5):

[0138]

[0139] Stage 2 Neural Differentiation Medium (Day 6-24):

[0140]

[0141] Stage 3 Neural Differentiation Medium (Days 25-43):

[0142]

[0143] Fluid exchange plan

[0144] DAY2-15: Change the medium every day.

[0145] DAY16-25: Change the medium every other day.

[0146] DAY26-43: Change medium every 2–3 days.

[0147] Cell morphology at each stage of culture Figure 2-9 shown.

[0148] In this example, two MSCs were obtained from the culture, one of which was obtained on the 21st day. The MSC flow cytometry identification chart is as follows: Fig. 9 As shown. Starting from around the 21st day, mesenchymal stem cells can be observed to be thrown out of the sphere and attached to the bottom of the 6-well plate. The hCO sphere is transferred to another new well, and 3 ml of NC misstion (Zhongsheng Tracing) medium is added to the well with mesenchymal stem cells to expand the mesenchymal stem cells. After full digestion (0.05%, room temperature digestion for 2 minutes), transfer to a 10 cm culture dish, add NC misstion (8 ml) and culture to obtain mesenchymal stem cells.

[0149] from Fig. 9 It can be seen that the cells do not express CD34, CD31 and CD45, but express CD73, CD105 and CD90, which is consistent with the international definition of mesenchymal stem cell surface markers. At the same time, they can differentiate into osteoblasts, chondrocytes and adipocytes, and are therefore defined as mesenchymal stem cells.

[0150] The other strain was obtained from digested hCO spheres on the 43rd day. The flow cytometry results were as follows: Fig.10 On the 43rd day, trypsin was used for digestion (0.5 ml, concentration 0.25%, digestion for 2 minutes at room temperature, blowing to disperse the hCO spheres, and after the clumps settled, the supernatant was discarded and 1 ml of NC misstion was added to resuspend), and then transferred to a 10 cm culture dish and NC misstion was added

[0151] (8ml) to induce mesenchymal stem cells.

[0152] from Fig.10 It can be seen that it does not express CD34, CD31 and CD45, but expresses CD73, CD105 and CD90, which meets the definition of international mesenchymal stem cell surface markers. At the same time, it can differentiate into osteoblasts, chondrocytes and adipocytes, and is therefore defined as mesenchymal stem cells.

[0153] Furthermore, we also found that pMSCs (especially pMSCs obtained by digesting hCO spheres on day 43, unless otherwise specified, all pMSCs used below are pMSCs obtained by digesting hCO spheres on day 43) have the ability to support the expansion of hematopoietic stem cells.

[0154] After long-term co-culture of hematopoietic stem and progenitor cells from umbilical cord blood, the number of hematopoietic stem and progenitor cells increased significantly (CD). CFU analysis showed that both hematopoietic stem cells (Mix) and progenitor cells (GM, G, M, BFU-e, CFU-e) increased significantly. Fig.11 shown.

[0155] Example 2: Cell Identification pMSCs have mesenchymal stem cell-specific markers (CD73, CD105, CD44, CD29CD90 positive; CD11b, CD34, CD45, CD144 negative)

[0156] 2.1 Mesenchymal stem cells derived from human brain organoid culture have mesenchymal stem cell-specific markers and neurotransmitters Characteristics

[0157] According to the method described in Example 1, we obtained human mesenchymal stem cells, i.e., pMSCs, from H1-hESC cells and human induced pluripotent stem cell (hiPSC) forebrain organoids. The brain-specific organoids generated from H1-hESC or hiPSC cells were digested with trypsin to produce a single cell suspension, which was transferred to a standard tissue culture plate in MSC culture medium. During repeated passages, the adherent cells gradually developed a spindle-shaped morphology until a uniform population was formed within a few weeks ( Fig.12 ). Based on the fact that this group of MSC-like cells is derived from the neural crest cell line of hPSC, we named this group of cells pMSC. This pMSC can differentiate into classic mesenchymal-derived cells (osteoblasts, chondrocytes and adipocytes) ( Fig.13 Flow cytometric analysis showed that for UC-MSCs, the vast majority of adherent pMSC cells expressed positive MSC markers (CD73, CD105, CD44, and CD29>99.8%, CD90>94.2%), whereas negative MSC markers, including CD11b, CD34, CD45, and CD144, were expressed in only a small fraction of these cells (<1%, Fig.14 , Fig.15 pMSC and UC-MSC grow rapidly in the early stage of passage, but their proliferation potential slows down slightly after several generations, and still tends to be in a stable proliferation period at P14-P20 ( Fig.16 ), unlike UC-MSCs, which rapidly enter the decay phase (aging or differentiation into fibroblasts) after a large number of proliferations in the early stage. In general, there was no significant difference in cell survival rate between pMSCs and UC-MSCs (≥95.5%, Fig.17 A).

[0158] In principal component analysis, pMSC and UC-MSC were separated from hPSC based on PC1 and PC2, and H1-pMSC and hiPSC-pMSC were placed next to each other for classification comparison ( Fig.17 B). RNA-seq analysis showed that in pMSCs and UC-MSCs, the expression of multipotent genes was significantly reduced, and the expression of MSC marker genes, adipogenic genes, and osteochondrogenic progenitor genes was significantly increased, indicating that pMSCs are essentially MSCs and have tri-directional differentiation potential ( Fig.18 , Fig.19 ). Compared with UC-MSC, H1-pMSC and hiPSC-pMSC had 1041 genes upregulated. Gene ontology analysis showed that the upregulated genes were related to axon development, mesenchymal development and neural crest cell differentiation ( Fig. 20 , Fig.21 CD271, a neural crest marker, was also rapidly lost after exposure to MSC culture medium, while MSC markers such as CD73 also aggregated in passaged pMSCs to levels similar to those of UC-MSCs ( Fig. 22 ). These data suggest that we have developed an efficient and safe protocol to derive pMSCs from human brain organoids with neural crest origin.

[0159] 2.2pMSCs support the expansion of human CB-HSPCs in long-term in vitro culture

[0160] To test the mesenchymal function in vitro, the ability of pMSCs to support HSPCs was evaluated by co-culturing umbilical cord blood (UCB)-derived CD34+ cells (UCB-CD34+ cells) on monolayers generated from each mesenchymal population. The effects of pMSCs on HSPCs were examined by FACS analysis and CFU assays. Fig.23 ). They were cultured in α-MEM medium without cytokines supplemented with low serum concentration (5%) or SFEM medium supplemented with SCF, TPO and FLT-3. In both media, the frequency and total number of CD34+Lin-HSPCs were significantly higher when co-cultured with pMSCs and UC-MSCs than in the absence of stromal cells ( Fig.24 When studying more primitive HSC-enriched populations of Lin-CD34+CD38- cells, the co-culture system was modified by the addition of exogenous growth factors to allow sufficient cell growth for analysis. In the presence of cytokines, the total number of cells co-cultured with pMSCs and UC-MSCs was significantly higher than that of uncultured stromal cells ( Fig.25 pMSC populations derived from H1 hESCs and hiPSCs were able to support CD34+CD38-Lin- cell growth for 1-5 weeks during co-culture ( Fig.26 , Fig. 27 ). When more HSPC subsets were analyzed, the results showed that the proportions or absolute numbers of CD34+CD38-CD90+CD45RA- long-term HSCs (LT-HSCs), CD34+CD38-CD90-CD45RA- short-term HSCs (ST-HSCs), and CD34+CD38-CD90-CD45RA+ committed progenitor cells (c-progenitor cells) were higher in the pMSC co-culture group (P<0.0001)( Fig.28 and 29 ).

[0161] Given that UCB HSPCs are multipotent and can differentiate into hematopoietic cell lineages with a variety of biological functions, we used hematopoietic CFU to test the colony-forming cell potential of UCB CD34+ cells under four culture conditions. Compared with UC-MSC co-culture or matrix-free culture, pMSC co-culture maintained significantly more CFU colonies ( Fig.30 A). A standard LTC-IC assay was established using irradiated pMSCs or UC-MSCs as feeder cells. After 5 weeks of co-culture, CB-CD34+ cells generated hematopoietic colonies on both feeder cell layers, and pMSCs had stronger in vitro stromal function than UC-MSCs ( Fig.30 B), indicating that pMSCs enhanced the overall differentiation potential of CB-CD34+ cells and maintained their multipotency.

[0162] Given that pMSCs are associated with increased proliferation rates of CD34+ cells, we further investigated whether pMSCs inhibit apoptosis and thus improve the viability of CD34+ cells. Annexin V / 7-AAD staining to detect apoptotic cells revealed that both early and late apoptosis rates of CB-CD34+ cells co-cultured with pMSCs and UC-MSCs were reduced ( Fig.31 ). Therefore, pMSCs and UC-MSCs could inhibit the apoptosis of CB-CD34+ cells compared with those without the addition of stromal cells.

[0163] 2.3pMSCs support the transplantability of human UCB-HSCs

[0164] In order to test the in vivo functional performance of HSCs maintained under in vitro culture conditions, 1×10 4 Fresh umbilical cord blood total nucleated cells with CD34+ cells were co-cultured with UC-MSCs, pMSCs, or without stromal cells for 2 weeks, and then the co-cultured total cells were transplanted into sublethally irradiated immunodeficient BNDG mice ( Fig.32 ). Flow cytometry was used to analyze the chimerism of human blood cells in the peripheral blood or bone marrow of recipient mice 3 or 6 months after transplantation. The results showed that at 3 months, the chimerism rate of pMSC co-cultured cells in the peripheral blood of mice was significantly higher than that of cells co-cultured without stromal cells or UC-MSC ( Fig.33 At 6 months, animals receiving pMSC coculture also had significantly higher levels of engraftment in the mouse BM compared with animals without stromal cells or UC-MSC coculture ( Fig.34 and 35 Reconstitution of multiple lineage hematopoietic cells, including CD3+ lymphocytes and CD33+ myeloid cells, was detected in the bone marrow of all mice treated with hematopoietic cells ( Fig.36). Taken together, these data demonstrate that pMSCs are able to maintain engraftable HSCs in vitro and reconstitute hematopoiesis in vivo, expanding and differentiating into HSPCs capable of downstream differentiation into functional mature blood cells.

[0165] 2.4 Co-culture of HSPC and pMSC can improve the microenvironment maintained by HSC

[0166] To investigate the role of pMSCs in preserving HSPCs, we compared transcriptome sequencing results of pMSCs and UC-MSCs using RNA-seq analysis. The expression of extracellular matrix genes, including collagen, fibronectin, laminin, and proteoglycans, was upregulated in both mesenchymal phenotypes ( Fig.37 ), which is important because the extracellular matrix (ECM) provides a structural scaffold that helps maintain HSC homeostasis (Domingues, Cao et al., 2017). The graphene oxide terms enriched in the upregulated genes in pMSCs included those regulating stem cell population maintenance, suggesting that pMSCs provide advantageous conditions for HSPC maintenance ( Fig.38 ). In addition, pMSCs expressed higher levels of MCAM (CD146); NES (NESTIN) (Sorrentino A, 2008; Acta Sinica, 2018; Pinho S, 2013); and HSC regulatory genes KITLG, JAG1, IGFBP2, and TNC than UC-MSCs (Costa et al., 2018; Huynh et al., 2008; Nakamura-Ishizu et al., 2012; Zhang et al., 2008) ( Fig.39 ). Compared with UC-MSCs, a total of 1047 genes were downregulated in H1-pMSCs or hiPSC-pMSCs ( Fig.40 A). GO terms enriched in these downregulated genes in pMSCs included regulation of stem cell differentiation, IL-6 production, cytokine production, and neutrophil migration, further supporting the hypothesis that pMSCs have an advantage in maintaining HSPCs ( Fig.40 B) Taken together, these findings suggest that pMSCs provide an improved microenvironment for HSC maintenance through a combination of mechanisms involving cell-cell contacts and secreted factors.

[0167] To understand the molecular mechanisms of how cells from different niches regulate UCB-CD34+ cells, we performed RNA sequencing analysis on CD34+ cells at day 7 after co-culture with or without pMSCs or UC-MSCs. Our principal component analysis (PCA) and Pearson distance tree data showed that CD34+ cells co-cultured with H1-pMSCs or hiPSC-pMSCs had similar transcriptome profiles to CD34+ cells co-cultured with UC-MSCs or without stromal cells ( Fig.40C). This indicates that H1-pMSC and hiPSC-pMSC have similar effects on UCB CD34+ cells. A total of 62 genes were upregulated after CD34+ cells were co-cultured with H1-pMSC or hiPSC-pMSC. Gene ontology analysis showed that genes related to extracellular matrix organization, collagen fibril organization, acidic chemical response and hyperoxia response, and Wnt signaling pathway regulation were upregulated ( Fig.41 ), indicating that CB-CD34+ and pMSC co-culture have significant advantages in resisting chemotherapy or external stimulation. Compared with not culturing stromal cells, a total of 171 genes were upregulated when CD34+ cells were co-cultured with H1-pMSC, hiPSC-pMSC and UC-MSC. These pMSC-specific upregulated genes are related to extracellular matrix organization, extracellular structure organization, cell-substrate adhesion, cytokine secretion, cell growth and cytokine secretion regulation ( Fig.42 ). In CD34+ cells co-cultured with UC-MSCs, a total of 794 genes were upregulated, and gene ontology analysis showed that most of them were genes related to neutrophil-mediated immunity, neutrophil activation, and positive regulation of interleukin-6 production ( Fig.42 ). In CD34+ cells co-cultured with pMSCs, the expression levels of progenitor cell characteristic genes such as CD38, CSF1R and ELANE were lower than those in cells co-cultured with UC-MSCs (Chen L, 2015). Interestingly, co-culture of CD34+ cells with pMSCs resulted in upregulation of the expression of HSC characteristic genes such as HOXA9, HLF and KIT, which is consistent with their known functions associated with the priming state ( Fig.43 ). In conclusion, these findings suggest that co-culture of UCB-CD34+ cells with pMSCs maintains the expression of genes characteristic of HSCs and enhances the ability to resist stimulation and interference from external inflammatory factors.

[0168] Example 3: pMSC specifically expresses markers that are not expressed by MSCs derived from iPSCs, umbilical cords, or bone marrow derived MSCs differentiated through other pathways

[0169] Furthermore, we sequenced multiple samples of pMSCs and sent them to Tianjin Novogene for transcriptome sequencing. The results showed that (e.g. Fig.44 In addition to mesenchymal stem cell markers, pMSCs also highly expressed SFRP1 and SFRP2.

[0170] Further analysis showed that the pMSCs of the present invention also highly expressed CD24 and IL17RD.

[0171] Preferably, the pMSCs of the present invention further highly express one or more characteristic functional molecules among COLEC12, FOXC1 and TGA8.

[0172] Furthermore, pMSCs also expressed high levels of NPTX1, NES, CXCL12, NFATC4, SIX1, CLSTN2, SCG2, MSX2, MDFI, TFAP2A, APOE, BMF, CPXM1, NYNRIN, DSP, CPE, PCDHB5, SNED1, AUTS2, DLX1, SESN3, AFF3, PALM, SENP7, FREM2, PCDHB2, ADAMTS10, TFAP2B, KIAA1755, DLX2, IGSF3, SHC2, TSPAN11, PLXNA4, TMEM246, VANGL2, and FAM131B.

[0173] Based on the results of RNA-seq analysis of MSC and CB-CD34+ cell co-culture, proteomic sequencing analysis was further performed on the supernatant of pMSC and UC-MSC culture fluid. In the entire RNA-seq dataset, most genes involved in the negative regulation of the Wnt signaling pathway were upregulated in pMSC compared with UC-MSC, including SFRP1, SFRP2, and IGFBP2 ( Fig.45 A). The gene with the greatest expression change between pMSCs and UC-MSCs was the Wnt antagonist secreted frizzled-related protein 1 (SFRP1) ( Fig.45 B). The expression of other Wnt inhibitors was also significantly increased in pMSC stroma, including SFRP2 and IGFBP2 ( Fig.45 B).

[0174] Proteomic sequencing analysis of the supernatants of pMSC and UC-MSC culture fluids showed that compared with UC-MSC, a total of 14 proteins were significantly upregulated in the supernatants of H1-pMSC or hiPSC-pMSC culture fluids ( Fig.46 ). The enriched GO terms for upregulated proteins in pMSCs included regulation of insulin-like growth factor receptor signaling pathway and negative regulation of Wnt signaling pathway ( Fig.46 ). Compared with UC-MSCs, 7 proteins and genes were upregulated in H1-pMSCs or hiPSC-pMSCs ( Fig.47 A). Compared with UC-MSCs, CB-CD34+ and pMSCs derived from two hPSCs showed increased expression of SFRP1 and SFRP2 ( Fig.47B), among which, the differences in SFRP1 were 6.35 times and 5.75 times (p value < 0.01); the differences in SFRP2 were 7.70 times and 8.03 times (p value < 0.01). Therefore, pMSCs express higher levels of Wnt inhibitors SFRP1, SFRP2, and IGFBP2 than UC-MSCs. This shows that pMSCs have a stronger ability to inhibit myeloid differentiation and resist inflammatory stimulation, thereby effectively maintaining the function of HSCs.

[0175] Example 4: pMSCs may promote the transplantability of hematopoietic stem cells by regulating cell cycle progression through antagonizing the Wnt signaling pathway.

[0176] To fully understand the purpose of Wnt inhibitors in pMSCs to change the expression of UCB-CD34+ cells, we compared the effects of various MSC co-cultures on the maintenance of CD34+ cells by transwell culture with the Wnt activator CHIR99021 (CHIR) or DMSO ( Fig.48 ). Addition of Wnt agonists promoted the rapid differentiation of Lin+ cells in pMSC and UC-MSC co-culture, resulting in the loss of CD34+Lin- and CD34+CD38-Lin- cells ( Fig.49 and 50 ). The latter observation is consistent with previous findings that canonical Wnt signaling regulates hematopoiesis in a dose-dependent manner, where high Wnt activation is detrimental to HSC self-renewal and leads to depletion of the LT-HSC pool (Famili et al., 2016; Luis et al., 2011). Interestingly, the proportion and number of CD34+Lin- and CD34+CD38-Lin- cells in pMSC co-cultures remained greater than those in UC-MSC co-cultures and CHIR without stromal cells ( Fig.49 and 50 This may be because the Wnt inhibitor secreted by pMSCs resisted the effect of CHIR on HSPC differentiation during transwell culture.

[0177] To determine whether pMSCs affect HSPC apoptosis and / or cell cycle, we used flow cytometry to analyze apoptosis of CD34+Lin-HSPCs on day 7 of transwell culture. The results showed that co-culture of CD34+Lin- cells with pMSCs significantly reduced the percentage of early apoptotic cells and significantly increased the percentage of viable cells ( Fig.51). 5'-Bromo-2'-deoxyuridine (BrdU) incorporation analysis showed that the cell cycle of 34+Lin- cells co-cultured with pMSCs and UC-MSCs was different. Under pMSC treatment, the reduced differentiation of CD34+Lin- cells was due to the induction of quiescence, while the Wnt activator CHIR promoted the entry of non-co-cultured 34+Lin- cells into the S phase, resulting in the loss of stemness of HSCs and differentiation into myeloid lineages ( Fig.52 ).

[0178] Taken together, all these data indicate that pMSCs express high levels of Wnt inhibitors, including SFRP1, SFRP2, and IGFBP2. When exposed to CHIR, pMSCs inhibit apoptosis and increase quiescence of HSPCs. pMSCs may maintain the transplantability of human HSCs by inhibiting Wnt signaling and regulating the cell cycle. These findings suggest that CB-CD34+ cells co-cultured with pMSCs have the necessary self-renewal potential to provide long-term multipotent reconstitution and resist external stimuli, in part by inhibiting the Wnt signaling pathway.

[0179] Other methods and steps:

[0180] 1. Isolation and culture of hUC-MSCs

[0181] Human umbilical cords (hUCs) without pregnancy or delivery complications were collected from West China Women and Children's Hospital and transferred into sterile boxes containing cold Hanks' balanced salt solution (HBSS) (Life Technologies, USA). Written informed consent was obtained from pregnant women before delivery. This study was approved by the Institutional Ethics Committee of the Institute of Transfusion Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS & PUMC).

[0182] The human umbilical cord was dissected longitudinally and the artery and vein were removed. The remaining part was cut into 0.2 cm3 sections (Zou et al., 2016). These explants were transferred to CELLstart TM CTS TMSubstrate (Life Technologies, USA) was coated on 100 mm plates (Nunc, Denmark). StemPro1 MSCSFM XenoFree medium (Life Technologies, USA) containing 1% penicillin-streptomycin (Life Technologies, USA) was added to the culture dish, and the explants were placed in a 5% CO2 incubator at 37°C without disturbance to allow cells to migrate from the explants. After 7-9 days, MSC-like cells were visible around the debris. The cells were passaged to another plate and when the cells reached 80% confluence, they were split 1:4 using 0.05% Trypsin-EDTA (Life Technologies, USA).

[0183] 2. In vitro adipogenic, chondrogenic and osteogenic differentiation

[0184] hiPSC-MSC differentiation was induced in different differentiation media for 21 days. In a six-well plate (TPP), 104 cells were seeded per well. In order to induce osteogenic differentiation, cells were cultured in a mesenchymal stem cell culture medium containing 1M dexamethasone, 0.5M ascorbic acid and 10mM b-glycerophosphate (all from Sigma-Aldrich). In order to induce adipogenesis, cells were cultured in MSC culture medium supplemented with 50g / mL indomethacin (Sigma-Aldrich), 50g / mL ascorbic acid and 100nM dexamethasone. In order to perform chondrogenic differentiation, hiPSC-MSCs were centrifuged in 0.2mL culture medium at a concentration of 500xg for 10 minutes in a 15mL Falcon tube to form a pellet. Microspheres were cultured in MSC culture medium supplemented with 0.01 M dexamethasone, 397 g / mL ascorbic acid-2-phosphate (Sigma-Aldrich), 1 mM sodium pyruvate (Sigma-Aldrich), 10 ng / mL transforming growth factor-1 (TGF-1, Life Technologies), and 1% insulin-transferrin-selenium (Life Technologies). Osteogenesis was assessed by alizarin red staining, adipogenesis by oil red staining, and chondrogenesis by alcian blue staining (all from Sigma-Aldrich).

[0185] 3. Hematopoietic colony assay

[0186] The hematopoietic potential of the cells was assessed by culturing on methylcellulose (H4320, STEMCELL) supplemented with 1% penicillin / streptomycin (Invitrogen) and cytokines as described previously (Ma et al., 2008). BFU-E, CFU-Mix, CFU-GM, CFU-G, and CFU-M colonies were assessed after 10-16 days.

[0187] 4. Long-term cultured hematopoietic initiating cells (LTC-IC) assay

[0188] The standard LTC-IC method was used to evaluate the in vitro matrix support capacity of UC-MSCs and pMSCs (Tormin, Li et al., 2011). Briefly, 5×10 3 Mesenchymal stem cells were plated in gelatin-coated 96-well culture dishes and irradiated (15 Gy) the next day. Primary human umbilical cord blood CD34+ cells were isolated from donor umbilical cord blood using magnetic-activated cell sorting. 24 hours after irradiation, 1000 UCB-CD34+ cells were selected and inoculated in SFEM medium containing 50 ng / ml SCF, 30 ng / ml Flt3 and 30 ng / ml TPO, and half of the medium was replaced every 3 days. After 6 weeks, cells were collected by trypsinization and hematopoietic colony formation was detected.

[0189] 5. Hematopoietic stem cell transplantation

[0190] Mice were housed in the SPF animal facility of the Stem Cell Research and Application Center of the Institute of Blood Transfusion. All animal experiments were approved by the Institutional Ethics Review Committee of the Institute of Blood Transfusion (IERC-IBT). Adult BNDG mice were purchased from Beijing Biocytogen Biotechnology Co., Ltd. 4 Human cord blood (CB) nucleated cells containing CD34+ cells were co-cultured with stromal cells for 2 weeks and then transplanted into irradiated (1.0 Gy) 8-10 week old BNDG mice via the retro-orbital sinus. Transplanted mice were maintained with water containing gentamicin sulfate for 2 weeks. Peripheral blood was obtained from the retro-orbital sinus. Flow cytometry analysis was performed 3 months after transplantation and 6 months after transplantation. CB nucleated cells were cultured in StemSpanTM SFEMII (Stemcell Technologies, Catalog No. 5. No. 09655) containing 50ng / ml SCF, 10ng / ml IL-11, 25ng / ml Flt3 and 5ng / ml TPO.

[0191] 6. Flow cytometry and cell sorting

[0192] Co-cultured cells were dissociated with 0.25% trypsin-EDTA solution (Invitrogen) and filtered through a 70 μm nylon mesh to obtain a single cell suspension. Flow cytometry was performed using a FACSCanto II system (BD Biosciences), and data analysis was performed using FlowJo software (v10.0.8.). Cell sorting was performed using a MoFloastrio high-speed cell sorter (Beckman Coulter). The antibodies used are shown in Table 1.

[0193] Table 1

[0194]

[0195] 7. Cell cycle analysis

[0196] Cells were treated with BrdU for 6 h, stained for surface antigens, and processed using the APC-BrdU FlowKit (BD) according to the manufacturer's instructions.

[0197] 8. Cell apoptosis detection

[0198] Apoptotic cells were stained with annexin V-APC and 7-AAD according to the manufacturer's instructions (BioLegend, catalog number: 640930). Apoptosis was detected using the FACSCanto II system (BD Biosciences).

[0199] 9. Statistical analysis

[0200] All data are expressed as mean ± SD. Statistical analysis was performed using t-test. P < 0.05 was considered statistically significant.

[0201] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A mesenchymal stem cell derived from a brain organoid, characterized in that: The mesenchymal stem cells have the following characteristics: (i) >94% of the cells express CD73, CD105, CD44, CD29, and CD90; (ii) <1% of the cells express CD11b, CD34, CD45, and CD144; and (iii) compared with mesenchymal stem cells from other sources, they express higher levels of SFRP1.

2. The mesenchymal stem cell according to claim 1, characterized in that <1% of the mesenchymal stem cells expressed CD271. Preferably, >99.8% of the mesenchymal stem cells express CD73, CD105, CD44 and CD29, Preferably, >94.2% of the mesenchymal stem cells express CD90. Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express a higher level of SFRP2. Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express a higher level of IGFBP2. Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of DKKL1, FRZB, SOX9, DACT3, SOX17, DKK3 and FRMD8, Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of at least one of CD24 and IL17RD, Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of COLEC12, FOXC1 and TGA8, Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of NPTX1, NES, CXCL12, NFATC4, SIX1, CLSTN2, SCG2, MSX2, MDFI, TFAP2A, APOE, BMF, CPXM1, NYNRIN, DSP, CPE, PCDHB5, SNED1, AUTS2, DLX1, SESN3, AFF3, PALM, SENP7, FREM2, PCDHB2, ADAMTS10, TFAP2B, KIAA1755, DLX2, IGSF3, SHC2, TSPAN11, PLXNA4, TMEM246, VANGL2 and FAM131B. Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of MCAM, NES, KITLG, JAG1, IGFBP2 and TNC, Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of CCDC126, SCG2, PSAP and COL18A1, Preferably, the mesenchymal stem cells express lower levels of genes involved in regulating stem cell differentiation, IL-6 production, cytokine production and neutrophil migration compared to mesenchymal stem cells from other sources.

3. A co-culture of brain organoid-derived mesenchymal stem cells and hematopoietic stem cells as described in claim 1 or 2.

4. The co-culture according to claim 3, characterized in that The mesenchymal stem cells support the expansion of the hematopoietic stem cells, maintain the transplantability of the hematopoietic stem cells, and improve the microenvironment maintained by the hematopoietic stem cells. Preferably, the hematopoietic stem cells include hematopoietic stem cells derived from umbilical cord blood, hematopoietic stem and progenitor cells derived from umbilical cord blood, and hematopoietic stem cells derived from bone marrow. Preferably, compared with the co-culture of mesenchymal stem cells and hematopoietic stem cells from other sources, the expression levels of CD38, CSF1R and ELANE progenitor cell characteristic genes in the hematopoietic stem cells are reduced, Preferably, compared with the co-culture of mesenchymal stem cells and hematopoietic stem cells from other sources, the expression levels of HOXA9, HLF and KIT HSC characteristic genes in the hematopoietic stem cells are increased, Preferably, the co-culture has more CD34+CD38-CD90+CD45RA- long-term HSCs, CD34+CD38-CD90-CD45RA- short-term HSCs and CD34+CD38-CD90-CD45RA+ committed progenitor cells compared to co-cultures of mesenchymal stem cells and hematopoietic stem cells from other sources.

5. A method for generating brain organoid-derived mesenchymal stem cells from human embryonic stem cells (hESC) or induced pluripotent stem cells (iPSC), characterized in that: The method comprises the following steps: (1) suspending hESCs or iPSCs in a Stage 0 medium containing Y-27632, and then culturing in a Stage 1 neural induction medium, a Stage 2 neural differentiation medium, and a Stage 3 neural differentiation medium in sequence, to generate brain organoids during such a culturing process, (2) deriving mesenchymal stem cells from the brain organoids, Wherein step (2) comprises: In the process of culturing brain organoids, adherent cells derived from brain organoids are cultured and expanded in MSC culture medium to obtain mesenchymal stem cells, or The generated brain organoids are dispersed into single cells, and the single cells of the brain organoids are cultured in an MSC culture medium, thereby obtaining mesenchymal stem cells.

6. The method according to claim 5, characterized in that In step (1), hESCs or iPSCs are cultured in a Stage 1 neural induction medium on days 2-5, in a Stage 2 neural differentiation medium on days 6-24, and in a Stage 3 neural differentiation medium on days 25-43, Preferably, the Stage 0 medium is ncTarget medium containing Y-27632, Preferably, the Stage 1 neural induction medium is a DMEM / F12 medium containing 10-20% (vol / vol) KSR, 1-5 μM Dorsomorphin and 5-10 μM SB-431542. Preferably, the Stage 2 neural induction medium is Neurobasal A medium containing 10-20 ng / ml EGF and 10-20 ng / ml FGF2. Preferably, the Stage 3 neural induction medium is Neurobasal A medium containing 10-20 ng / ml BDNF and 10-20 ng / ml NT3.

7. The method according to any one of claims 5-6, characterized in that The generated brain organoid-derived mesenchymal stem cells have the following characteristics: (i) >94% of cells express CD73, CD105, CD44, CD29, and CD90; (ii) <1% of cells express CD11b, CD34, CD45, and CD144; (iii) compared with mesenchymal stem cells from other sources, they express higher levels of SFRP1, Preferably, <1% of the mesenchymal stem cells express CD271. Preferably, >99.8% of the mesenchymal stem cells express CD73, CD105, CD44 and CD29, Preferably, >94.2% of the mesenchymal stem cells express CD90. Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express a higher level of SFRP2. Preferably, the generated brain organoid-derived mesenchymal stem cells also express higher levels of IGFBP2 compared to mesenchymal stem cells from other sources. Preferably, compared with mesenchymal stem cells from other sources, the mesenchymal stem cells also express higher levels of one or more of DKKL1, FRZB, SOX9, DACT3, SOX17, DKK3 and FRMD8, Preferably, the generated brain organoid-derived mesenchymal stem cells further express higher levels of at least one of CD24 and IL17RD compared to mesenchymal stem cells from other sources. Preferably, the generated brain organoid-derived mesenchymal stem cells also express higher levels of one or more of COLEC12, FOXC1 and TGA8 compared to mesenchymal stem cells from other sources, Preferably, compared with mesenchymal stem cells from other sources, the generated brain organoid-derived mesenchymal stem cells also express higher levels of NPTX1, NES, CXCL12, NFATC4, SIX1, CLSTN2, SCG2, MSX2, MDFI, TFAP2A, APOE, BMF, CPXM1, NYNRIN, DSP, CPE, PCDHB5, SNED1, AUTS2, DLX1, SESN3, AFF3, PALM, SENP7, FREM2, PCDHB2, ADAMTS10, TFAP2B, KIAA1755, DLX2, IGSF3, SHC2, TSPAN11, PLXNA4, TMEM246, VANGL2 and FAM131B. Preferably, compared with mesenchymal stem cells from other sources, the generated brain organoid-derived mesenchymal stem cells also express higher levels of one or more of MCAM, NES, KITLG, JAG1, IGFBP2 and TNC, Preferably, the generated brain organoid-derived mesenchymal stem cells also express higher levels of one or more of CCDC126, SCG2, PSAP and COL18A1 compared to mesenchymal stem cells from other sources. Preferably, the generated brain organoid-derived mesenchymal stem cells express lower levels of genes involved in regulating stem cell differentiation, IL-6 production, cytokine production, and neutrophil migration compared to mesenchymal stem cells from other sources, Preferably, the generated brain organoid-derived mesenchymal stem cells support the expansion of hematopoietic stem cells, maintain the transplantability of hematopoietic stem cells, and improve the microenvironment for hematopoietic stem cell maintenance.

8. Use of the brain organoid-derived mesenchymal stem cells as described in claim 1 or 2 for supporting the expansion of hematopoietic stem cells, maintaining the transplantability of hematopoietic stem cells, and improving the microenvironment for the maintenance of hematopoietic stem cells.

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