Methods of improving hematopoietic grafts

Abstract

The present invention relates to methods of improving hematopoietic grafts. In particular, the present invention relates to a method of preparing a hematopoietic cell graft or enriching hematopoietic stem cells capable of long term multilineage implantation and self-renewal from a population of cells. The invention also relates to hematopoietic grafts comprising said hematopoietic stem cells and their use in therapy.

Description

[0001] This application is a divisional application of the application with international application date of March 21, 2018, international application number PCT / EP2018 / 057197, entering the Chinese national phase on September 23, 2019, application number 201880020207.1, and invention name “Method for improving hematopoietic transplants”. Technical Field

[0002] The present invention relates to the field of medicine, and in particular to human hematopoietic transplants. Specifically, the present invention relates to the identification and selection of hematopoietic stem cells capable of long-term multi-lineage engraftment and self-renewal, ie, hematopoietic stem cells suitable for hematopoietic transplantation. Background Art

[0003] Hematopoietic stem cells (HSCs) are rare cells in human bone marrow (BM) and blood that are responsible for the lifelong curative effect of allogeneic hematopoietic cell transplantation in blood diseases or after radiotherapy / chemotherapy. These cells can be harvested from several sources including BM, mobilized peripheral blood or human umbilical cord blood. Umbilical cord blood provides several advantages, namely reduced demand for HLA matching and reduced risk of graft-versus-host disease. However, despite progress in the operation of HSCs, their number is still generally insufficient for allogeneic transplantation, and the resulting cells show lower multi-lineage and implantation potential compared to freshly isolated HSCs. On the basis of these findings, generating transplantable HSCs from non-hematopoietic sources appears to be one of the main goals in regenerative medicine.

[0004] A large number of protocols have been developed that utilize direct transformation of a variety of cell types, including cell fusion, reprogramming of differentiated cells caused by forced expression of transcription factors, or directed differentiation from human pluripotent stem cells (Wahlster and Daley, Nature cell biology, 2016, 18, 1111-1117). In many cases, the introduction of plasmids encoding oncogenes in recipient cells or the use of non-GMP grade feeder cells prevents them from being used in clinical applications. Finally, many of these protocols aim to produce cell populations with surface phenotypes similar to truly transplantable umbilical cord blood or adult HSCs, a strategy that has been shown to produce cells with poor implantation potential.

[0005] Therefore, there remains a need for products and methods to improve the efficiency of hematopoietic transplantation, including increasing the engraftment potential of transplanted cells and improving bone marrow replacement. Summary of the invention

[0006] The present invention aims to provide products and methods for improving the efficiency of hematopoietic transplantation. Specifically, the present invention provides methods for obtaining and selecting hematopoietic stem cells capable of long-term multi-lineage engraftment and self-renewal in vivo. The present invention paves the way for the use of pluripotent stem cells, especially induced pluripotent stem cells, as a source of HSC transplant cells.

[0007] Therefore, the present invention relates to an in vitro method for preparing a hematopoietic cell transplant or enriching hematopoietic stem cells capable of long-term multi-lineage engraftment and self-renewal from a cell population, the method comprising:

[0008] a) providing a cell population comprising hematopoietic stem cells, preferably early primitive hematopoietic stem cells, and

[0009] b) sorting cells of said population on the basis of expression of cell surface antigens CD135 and / or CD110, and

[0010] c) Recover CD135+ and / or CD110+ cells.

[0011] Preferably, in step b), cells are sorted based on the expression of the cell surface antigen CD110, and the cells recovered in step c) are CD110+. Optionally, in step b), cells can be further sorted based on the expression of the cell surface antigen CD135, and the cells recovered in step c) are CD110+CD135+.

[0012] The method may further comprise, before, after or simultaneously with step b), sorting cells based on the expression of apelin receptor (APLNR) and recovering APLNR+ cells.

[0013] The cell population provided in step a) may include hematopoietic stem cells obtained from peripheral blood, placental blood, umbilical cord blood, bone marrow, liver and / or spleen and / or may include immortalized hematopoietic stem cells.

[0014] Alternatively or additionally, the cell population provided in step a) may comprise hematopoietic stem cells obtained from in vitro differentiation of pluripotent stem cells, preferably induced pluripotent stem cells.

[0015] In some embodiments, the method may further include, before step a):

[0016] providing pluripotent stem cells, preferably induced pluripotent stem cells,

[0017] Induce embryoid body (EB) formation,

[0018] EBs are cultured in a liquid medium that primes pluripotent stem cells to differentiate into endo-hematopoietic lineages, and

[0019] Dissociate EB cells,

[0020] The cell population provided in step a) is thereby obtained.

[0021] Preferably, the liquid culture medium comprises stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 (FLT3) ligand, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1).

[0022] Preferably, the pluripotent stem cells are cultured in the liquid culture medium for 14 to 19 days, preferably 15 to 18 days, more preferably 17 days.

[0023] On the other hand, the present invention also relates to the use of CD135 and / or CD110 as a marker for hematopoietic stem cells capable of engraftment, in particular capable of long-term multi-lineage engraftment and self-renewal.

[0024] In another aspect, the present invention relates to a hematopoietic cell transplant comprising cells and a pharmaceutically acceptable carrier, wherein at least 10% of the cells are CD135+ and / or CD110+ hematopoietic stem cells. It also relates to a hematopoietic cell transplant prepared by the method of the present invention.

[0025] On the other hand, the present invention also relates to a hematopoietic cell transplant of the present invention, which is used to treat malignant diseases such as multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorder, chronic lymphocytic leukemia, juvenile chronic myeloid leukemia, neuroblastoma, ovarian cancer and germ cell tumors, or non-malignant diseases such as autoimmune disorders, amyloidosis, aplastic anemia, paroxysmal nocturnal hemoglobinuria, Fanconi'sanemia, Blackfan-Diamond anemia, major thalassemia, sickle cell anemia, severe combined immunodeficiency, Wiskott-Aldrich syndrome and inborn errors of metabolism.

[0026] The hematopoietic stem cell transplant can be used for autologous, syngeneic or allogeneic transplantation.

[0027] On the other hand, the present invention also relates to a liquid cell culture medium comprising (i) plasma, serum, platelet lysate and / or serum albumin, and (ii) transferrin or its substitute, insulin or its substitute, stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor (ESF). Growth factor 1 (IGF1), preferably relates to a liquid cell culture medium comprising (i) plasma, serum and / or platelet lysate, and (ii) transferrin, insulin, stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1).

[0028] Specifically, the liquid cell culture medium may contain:

[0029] - 10 to 100 ng / mL SCF, preferably 10 to 50 ng / mL SCF;

[0030] - 10 to 100 ng / mL TPO, preferably 10 to 50 ng / mL TPO;

[0031] - 100 to 500 ng / mL of FLT3-L, preferably 250 to 350 ng / mL of FLT3-L;

[0032] - 10 to 100 ng / mL of BMP4, preferably 10 to 50 ng / mL of BMP4;

[0033] - 50 to 300 ng / mL of VEGF, preferably 150 to 250 ng / mL of VEGF;

[0034] - 10 to 100 ng / mL IL3, preferably 20 to 80 ng / mL IL3;

[0035] - 10 to 100 ng / mL IL6, preferably 20 to 80 ng / mL IL6;

[0036] - 1 to 20 ng / mL IL1, preferably 1 to 10 ng / mL IL1;

[0037] - 10 to 200 ng / mL GCSF, preferably 50 to 150 ng / mL GCSF; and / or

[0038] - 10 to 150 ng / mL of IGF1, preferably 10 to 100 ng / mL of IGF1.

[0039] Preferably, the liquid cell culture medium comprises:

[0040] - 10 to 100 ng / mL SCF, preferably 10 to 50 ng / mL SCF;

[0041] - 10 to 100 ng / mL TPO, preferably 10 to 50 ng / mL TPO;

[0042] - 10 to 100 ng / mL of FLT3-L, preferably 10 to 50 ng / mL of FLT3-L;

[0043] - 50 to 300 ng / mL of BMP4, preferably 150 to 250 ng / mL of BMP4;

[0044] - 50 to 300 ng / mL of VEGF, preferably 150 to 250 ng / mL of VEGF;

[0045] - 10 to 100 ng / mL IL3, preferably 20 to 80 ng / mL IL3;

[0046] - 10 to 100 ng / mL IL6, preferably 20 to 80 ng / mL IL6;

[0047] - 1 to 20 ng / mL IL1, preferably 1 to 10 ng / mL IL1;

[0048] - 10 to 200 ng / mL GCSF, preferably 50 to 150 ng / mL GCSF; and / or

[0049] - 1 to 20 ng / mL of IGF1, preferably 1 to 10 ng / mL of IGF1.

[0050] The liquid culture medium may further comprise: (i) plasma, serum, platelet lysate and / or serum albumin, preferably plasma, serum and / or platelet lysate, and (ii) insulin or its substitute and transferrin or its substitute, preferably insulin and transferrin. Specifically, the liquid culture medium may further comprise:

[0051] - 1% to 20% plasma or serum, preferably 2% to 10% plasma or serum; or 0.1% to 2% platelet lysate, preferably 0.2% to 1% platelet lysate; and

[0052] - 5 µg / mL to 20 µg / mL, preferably 8 µg / mL to 12 µg / mL of insulin; and

[0053] - 10 µg / mL to 100µg / mL of transferrin, preferably 30 µg / mL to 60 µg / mL of transferrin.

[0054] The present invention also relates to the use of the liquid cell culture medium of the present invention for the growth and / or differentiation of cells of the hematopoietic lineage, for the differentiation of embryoid bodies, for the production of hematopoietic cell transplants. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 : Characterization of hiPSC-derived cells. (A) Experimental scheme. HiPSCs were differentiated into EBs over 17 days in the continuous presence of growth factors and cytokines. EB cells were characterized at different time points using q-PCR and flow cytometry. Images depict representative EBs at D13 and 17, respectively. (B) Hierarchical clustering outlining the expression of CD34 in EBs at D3, D7, D9, D13, D15, and D17. + Expression of a panel of 49 genes characterizing endothelial, hemogenic endothelial, and hematopoietic cells over time in cord blood cells. (C) Q-PCR patterns representing genes in the EHT equilibrium of D13 to D17 EB cell differentiation. For each gene, the fold change is the mean + / - SEM of 6 experiments. (D) Flow cytometric analysis of human CD309, ITGA2, MPL, and CKIT at D13 and D17 of EB culture. (E) Flow cytometric analysis of the expression of APLNR and CXCR4 at D7 to D17 of EB culture.

[0056] Figure 2 : Functional endothelial-hematopoietic profiling between D15 and D17. (A) In vitro assay probing the presence of endothelial (1-3) and hematopoietic (4-5) progenitors in EBs over time. Dissociated D15-17 EB cells give rise to: (1) CFC-ECs, (2) pseudo-microtubules, (3) EC-like cells capable of several passages, (4) CFCs, and (5) LTC-ICs. (B) Experimental scheme for in vivo assay probing the endothelial capacity of D16 cells. (C) Sections of D16 cell / hMSC plugs. Masson's trichrome stain. (D) Sections of D16 cell / hMSC plugs. Human von Willebrand factor +Cells (blue) immunostained. (E) D16 cell / hMSC plug sections, human CD31 + Cells (red).

[0057] Figure 3 : In vivo engraftment of D17 EB cells in immunocompromised NSG mice. (A) Experimental design. (B) Expression of human and mouse CD45 in primary recipients. + Comparison of representative flow cytometric analyses of cell engraftment. (C-D) hCD34 in the bone marrow of primary (C) or secondary (D) mice 20 weeks after transplantation. + hCD43 + hCD45 + Percentage of cells. Data are mean + / - SEM. (E) Distribution of human hematopoietic lineages in primary and secondary recipients. Numbers were normalized to 100%. (F) Colony formation assay of BM cells isolated from primary and secondary recipients. Frequency of CFU-GM, BFU-E, and CFU-GEMM colonies. (G) Representative colonies of CFU-GEMM (1), BFU-E (2), and CFU-GM (3) from primary and secondary BM recipients. (H) Cytospin. May Grünwald-Giemsa staining of cells isolated from colony formation assays performed on primary and secondary recipients. Mature macrophages (1), tissue monocytes (2), myeloid cells (2), and erythroblasts (3). (I) CB CD34 + Expression of human globin in erythroid cultures, BM from primary and secondary recipients, and BFU-E from BM from primary recipients. Data are mean + / - SEM. (J) Maturation of human T cells. hCD2 stained with antibodies against hTCR αβ and hTCR γδ + Peripheral blood sorted cells. (K) Functionality of human T cells. Whole thymic populations were CFSE labeled at D0 and expressed according to hCD3 + (Green) Gating. At D5, the unstimulated population is red, while the stimulated maternal population is blue.

[0058] Figure 4 :APLNR + Functional and molecular characterization of the population. (A) APLNR in the inoculum + The percentage of hCD45 cells in primary recipients of NOD-SCID BM at 18 weeks after transplantation + The correlation between the percentage of cells. (B) APNLR + (n=6, blue dots) and APNLR - (n=4, red dots) Engraftment capacity of the population. Cells with reconstructive potential were expressed in the APLNR +Data are presented as mean + / - SEM of percentage of human engraftment 18 weeks after transplantation. (C) APLNR using CD45, TIE, ENG, and CKIT anti-human antibodies + Combined flow cytometry analysis of populations. (D) PCA performed using a set of 49 mRNAs as variables and 6 cell populations as observations. Score plot of PC1 relative to PC2. The PC1 dimension may correspond to the trait "hematopoietic differentiation", which accounts for 44.9% of the variance. HiPSCs separate from the spindle. (E) PCA performed using a set of 49 mRNAs as variables and populations that are or are not endowed with transplant potential. The PC3 dimension, which accounts for 19.32% of the variance, separates the two groups. (F) Heat map of 8 genes that allow separation of the two groups.

[0059] Figure 5 :APLNR + and APLNR - Characterization of the population. Using D17 APLNR - (left side) or APLNR + Representative cross-sections of hCD45 and hCD43 expression in BM of mice transplanted with cell fractions.

[0060] Figure 6 : Unsupervised principal component analysis of the 5859 differential genes between the groups allowed to significantly distinguish the sample groups with a p-value of 4.75E-8 on the main plot.

[0061] Figure 7 : Circos plots depicting supervised analysis by significance analysis of microarrays (SAM) between each xenograft and HSC groups were performed to discover HSC biomarkers in each SRC-IPSC group.

[0062] Figure 8 : Venn diagram comparing HSC biomarkers enriched in each SRC-IPSC group shows any common genes.

[0063] Fig. 9 : Experimental design for in vivo engraftment of sorted D17 EB cells in immunocompromised NSG mice.

[0064] Fig.10 : At 20 weeks after transplantation, hCD34 + hCD43 + hCD45 + Data are mean + / - SEM. DETAILED DESCRIPTION OF THE INVENTION

[0066] The first transplantable HSCs are generated during embryonic development from a specialized population of endothelial cells (ECs) termed hemogenic endothelium. Following the endothelial-to-hematopoietic transition (EHT), these hemogenic ECs differentiate into hematopoietic cells (HCs), including HSCs, enter the circulation, expand in the embryonic liver, and arrive at their final residence, the BM. These early steps of developmental hematopoiesis, especially the generation of hemogenic ECs and budding of HCs, are fully recapitulated in embryoid body (EB) cultures.

[0067] Herein, the inventors developed a one-step, vector-free and matrix-free systematic procedure to direct the differentiation of human induced pluripotent stem cells (hiPSCs) into the endothelial-hematopoietic lineage. Although CD34+CD45+ progenitors emerge from bursting EBs at day 10 (D) until day 14 in the standard protocol, the culture conditions used by the inventors provided D17 embryoid bodies that exhibited well-defined compact spherical structures and no bursting, thus evaluating as a dramatic delay in the differentiation process. These culture conditions were applied to three different hiPSC cell lines with different reprogramming protocols, such as using episomes or retroviruses, and similar differentiation efficacy was obtained, thus confirming the robustness of the method.

[0068] Based on the analysis of these differentiated embryoid body cells and the bioinformatics analysis of the transcriptome of hematopoietic stem cells that can only implant at the primary level or can implant at the primary level and at the secondary level, the inventors here identified a sub-fraction of early primitive hematopoietic stem cells that not only exhibited high implantation capacity, but also exhibited a strong and durable self-renewal capacity, making these cells an ideal source for hematopoietic transplantation. They found that this sub-fraction can be characterized by the expression of Fms-like tyrosine kinase 3 receptor (FLT3 or CD135) and / or thrombopoietin receptor (MPL or CD110) and / or apelin receptor (APLNR).

[0069] Therefore, in a first aspect, the present invention relates to a method for preparing a hematopoietic cell transplant, preferably an in vitro method, the method comprising:

[0070] a) providing a cell population comprising hematopoietic stem cells, and

[0071] b) sorting the cells of said population on the basis of the expression of the cell surface antigen CD135 and / or CD110 and / or apelin receptor (APLNR), preferably on the basis of the cell surface antigen CD110, and

[0072] c) recovering CD135+ and / or CD110+ and / or APLNR+ cells, preferably CD110+ cells.

[0073] The present invention also relates to a method, preferably an in vitro method, of enriching hematopoietic stem cells suitable for hematopoietic transplantation, i.e. capable of long-term multi-lineage engraftment and self-renewal, from a cell population, the method comprising:

[0074] a) providing a cell population comprising hematopoietic stem cells, and

[0075] b) sorting the cells of said population on the basis of the expression of cell surface antigens CD135 and / or CD110 and / or apelin receptor (APLNR), preferably on the basis of the cell surface antigen CD110, and

[0076] c) recovering CD135+, CD110+ and / or APLNR+ cells, preferably CD110+ cells.

[0077] The recovered CD135+ and / or CD110+ and / or APLNR+ cells can be used as a hematopoietic transplant, or can be included in or added to a hematopoietic transplant (eg, a bone marrow or umbilical cord blood transplant) in order to improve the efficacy of the transplant.

[0078] As used herein, the term "CD135" or "FLT3" refers to a class III receptor tyrosine kinase activated by the binding of the cytokine Flt3 ligand (FLT3L) to the extracellular domain. In humans, the gene is encoded by the FLT3 gene (Gene ID: 2322). Upon activation, CD135 phosphorylates and activates a variety of cytoplasmic effector molecules involved in the pathways of apoptosis, proliferation, and differentiation of hematopoietic cells in the bone marrow. Mutations that lead to constitutive activation of this receptor cause acute myeloid leukemia and acute lymphoblastic leukemia.

[0079] As used herein, the term "CD110" or "MPL" refers to the thrombopoietin receptor, also known as myeloproliferative leukemia protein. In humans, CD110 is encoded by the MPL (myeloproliferative leukemia virus) oncogene (Gene ID: 4352). CD110 is a 635 amino acid transmembrane domain with two extracellular cytokine receptor domains and two intracellular cytokine receptor box motifs. Its ligand, thrombopoietin, has been shown to be a major regulator of megakaryocyte production and platelet formation.

[0080] As used herein, the term "APLNR" refers to the apelin receptor, a G protein-coupled receptor that binds apelin. This receptor has been shown to be involved in the cardiovascular and central nervous systems, in glucose metabolism, in embryonic and tumor angiogenesis, and as a human immunodeficiency virus coreceptor. In humans, this receptor is encoded by the APLNR gene (Gene ID: 187).

[0081] As used herein, the term "hematopoietic cell transplant" or "hematopoietic graft" refers to an ex vivo cell product for hematopoietic transplantation. The hematopoietic cell transplant may comprise hematopoietic stem cells obtained from mobilized peripheral blood, placental blood, umbilical cord blood, amniotic fluid, bone marrow, liver and / or spleen, as well as immortalized HSCs and / or HSCs obtained from differentiation of pluripotent stem cells (e.g., induced pluripotent stem cells).

[0082] The cell population provided in step a) comprises hematopoietic stem cells (HSCs), in particular early primitive HSCs.

[0083] Preferably, the cell population provided in step a) is a population of human cells.

[0084] As used herein, the term "hematopoietic stem cell" or "HSC" refers to a cell with both multipotency and self-renewal capabilities. Multipotency is the ability to differentiate into all functional blood cells such as B cells, T cells, NK cells, lymphoid dendritic cells, myeloid dendritic cells, granulocytes, macrophages, megakaryocytes, and erythroid cells. Self-renewal is the ability to generate HSC itself without differentiation.

[0085] When used herein, the term "early primitive HSC" refers to an HSC that is a precursor of CD34+ / CD45+ HSC and has both multipotency and self-renewal capabilities. Early primitive HSC belongs to the hemogenic endothelium that can undergo endothelial to hematopoietic transition and can be CD34- / CD45- or CD34+ / CD45-. Early primitive HSC may also express CXCR4 and / or show upregulation of genes involved in early hematopoietic commitment (e.g., HOXB4, c-MYC, and MITF), self-renewal (e.g., HOXA9, ERG, and RORA), and stemness (e.g., SOX4 and MYB), and / or may be a long-term culture initiating cell (LTC-IC), i.e., an HSC that can generate colony-forming unit cells (CFU) after 5 to 8 weeks (35 to 60 days) of culture on a bone marrow (BM) matrix (Miller and Eaves, Methods Mol Med. 2002; 63: 123-41). In certain preferred embodiments, the term "early primitive HSC" refers to CD34- / CD45- or CD34+ / CD45- LTC-IC cells. In certain other embodiments, the term "early primitive HSC" may also refer to CD34+ / CD45+ or CD34- / CD45+ LTC-IC cells.

[0086] The cell population provided in step a) may comprise HSCs, immortalized HSCs and / or pluripotent stem cells obtained from peripheral blood, placental blood, umbilical cord blood, amniotic fluid, bone marrow, liver and / or spleen.

[0087] In an embodiment, the cell population provided in step a) comprises cells obtained from peripheral blood, placental blood, umbilical cord blood, amniotic fluid, bone marrow, liver and / or spleen, preferably cells obtained from peripheral blood, placental blood, umbilical cord blood and / or bone marrow, or consists of said cells. Specifically, the cell population provided in step a) may be a cell population obtained from peripheral blood, placental blood, umbilical cord blood, amniotic fluid, bone marrow, liver or spleen, preferably a cell population obtained from peripheral blood, placental blood, umbilical cord blood or bone marrow.

[0088] HSCs may be obtained from the various sources mentioned above using any method known to the skilled person.

[0089] For example, peripheral blood stem cells can be present in a whole blood sample, or can be collected from the blood by a method known as apheresis. The yield of peripheral blood stem cells can be increased by administering compounds that stimulate the migration of stem cells from the donor's bone marrow into the peripheral circulation. These compounds include, for example, granulocyte colony stimulating factor or Mozobil™ (plerixafor). After this treatment, peripheral blood is often referred to as "mobilized peripheral blood."

[0090] HSCs can also be obtained from the bone marrow of a subject. In this case, HSCs are removed from a large bone of the subject, usually the pelvic bone, through a large needle that reaches into the center of the bone.

[0091] Umbilical cord blood or placental blood, which can be obtained when a mother donates her baby's umbilical cord or placenta after giving birth, has a higher concentration of HSCs than is normally found in adult blood.

[0092] In a more specific embodiment, the cell population provided in step a) is a sample of peripheral blood, preferably mobilized peripheral blood, bone marrow, umbilical cord blood or placental blood.

[0093] In another embodiment, the cell population provided in step a) comprises or consists of immortalized HSCs, preferably human immortalized HSCs. HSCs can be immortalized using any method known to the skilled person, such as retroviral-mediated gene transfer of β-catenin (Templin et al., Exp Hematol. 2008 Feb;36(2):204-15).

[0094] In another embodiment, the cell population provided in step a) comprises or consists of HSCs obtained by in vitro differentiation of pluripotent stem cells, and the pluripotent stem cells are preferably induced pluripotent stem cells.

[0095] In a preferred embodiment, the cell population provided in step a) comprises or consists of HSCs obtained from differentiation of induced pluripotent stem cells (iPSCs), preferably from differentiation of human iPSCs.

[0096] iPSCs are derived from non-pluripotent cells, usually adult somatic cells, by a process known as reprogramming, in which only a few specific genes need to be introduced to confer pluripotency to the cells. Various gene combinations have been shown to confer pluripotency to the cells, such as Oct4 / Sox2 / Nanog / Lin28 or Oct4 / Sox2 / KLF / cMyc. One benefit of using iPSCs is that the use of embryonic cells is completely avoided, and therefore any ethical issues thereof are avoided.

[0097] iPSCs may be obtained from the subject to be treated (transplant patient) or from another subject. Preferably, iPSCs are derived from cells from the subject to be treated, in particular from fibroblasts of the subject.

[0098] Pluripotent stem cells, and in particular iPSCs, can be differentiated into HSCs, or more specifically into early primitive HSCs, using any method known to the skilled person, for example using any of the methods described in Bathia (supra), Doulatov et al. (Cell Stem Cell. 2013 Oct 3; 13(4): 10.1016), or Sandler et al. (Nature. 2014 Jul 17; 511(7509):312-8).

[0099] In a specific embodiment, the method of the present invention further comprises, before step a):

[0100] Providing pluripotent stem cells, in particular iPSCs, preferably human iPSCs,

[0101] Induce embryoid body (EB) formation,

[0102] culturing the EBs in a liquid medium that initiates differentiation of the pluripotent stem cells into endothelial-hematopoietic lineages, and

[0103] Dissociate EB cells,

[0104] Thereby the cell population provided in step a) of the method of the invention and as described above is obtained.

[0105] Embryoid body formation from pluripotent stem cells can be obtained by any protocol known to the skilled person. For example, pluripotent stem cells can be treated with collagenase IV and transferred to low attachment plates in liquid culture medium.

[0106] The differentiation of the pluripotent stem cells into the endothelial-hematopoietic lineage is then obtained by culturing the embryoid bodies in a liquid medium that induces the differentiation. This liquid medium may be the same as the medium used during the formation of the embryoid bodies.

[0107] Several culture media that induce differentiation of pluripotent stem cells toward the endothelial-hematopoietic lineage have been described (see, for example, Lapillonne et al., Haematological, 2010; 95(10), Doulatov et al., Cell Stem Cell. 2013, 13(4)), and they can be used in the present invention.

[0108] However, the inventors have found that a culture medium containing a specific combination of cytokines and growth factors provides differentiated embryoid bodies that exhibit well-defined, compact spherical structures without bursts. Therefore, in a specific embodiment, the culture medium that initiates differentiation of pluripotent stem cells into the endothelial-hematopoietic lineage comprises stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 (FLT3) ligand, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1). This culture medium may also contain plasma, serum, platelet lysate, serum albumin, transferrin or a substitute thereof and / or insulin or a substitute thereof, preferably (i) plasma, serum and / or platelet lysate, and (ii) transferrin and insulin.

[0109] In a preferred embodiment, the culture medium that initiates differentiation of pluripotent stem cells toward the endothelial-hematopoietic lineage is a culture medium of the present invention and is described hereinafter.

[0110] Preferably, the embryoid bodies are cultured in the liquid culture medium for 14 to 19 days, more preferably 15 to 18 days, even more preferably 17 days. In a preferred embodiment, the embryoid bodies are cultured in the liquid culture medium of the present invention and described hereinafter for 14 to 19 days, more preferably 15 to 18 days, even more preferably 17 days.

[0111] The differentiated embryoid bodies are then dissociated, for example by incubation with collagenase B and cell dissociation buffer or using any other method known to the skilled person.

[0112] As demonstrated in the experimental part of the present application, the population of dissociated cells comprises HSCs, in particular early primitive HSCs, and can be provided in step a) of the method of the present invention.

[0113] The presence of early primitive HSCs in a population of HSC-containing cells can be assessed by any method known to those skilled in the art, such as using the long-term culture initiating cell (LTC-IC) assay described in Liu et al., Methods Mol Biol. 2013;946:241-56.

[0114] Prior to use in the methods of the invention, HSCs, including early primitive HSCs, may be stored. Specifically, the cells may be cryopreserved for a long term, optionally in the presence of a cryoprotectant such as DMSO.

[0115] In step b) of the method of the present invention, the cells of the population provided in step a) are sorted based on the expression of cell surface antigens CD135 and / or CD110 and / or the expression of APLNR.

[0116] When used in this article, the term "sorting" of cells refers to the operation of grouping cells according to specified criteria, such as marker expression. Any method known to professionals to separate cells according to specified criteria can be used, including but not limited to fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS). When used in this article, the expression "sorting on the basis of the expression of a specific protein, such as a cell surface antigen" refers to the operation of separating cells that express the protein and cells that do not express the protein. In a preferred embodiment, the expression of CD135, CD110 or APLNR is detected at the cell surface. However, any other method known to professionals and allowing the detection of such expression, such as a method for detecting a specific mRNA (e.g., RT-PCR), can be used.

[0117] Cells can be sorted based on:

[0118] - expression of the cell surface antigens CD135 and CD110, and optionally expression of APLNR; or

[0119] - expression of the cell surface antigen CD135, and optionally APLNR and CD110; or

[0120] - expression of the cell surface antigen CD135, and optionally expression of APLNR; or

[0121] - expression of the cell surface antigen CD135, and optionally CD110; or

[0122] - expression of the cell surface antigen CD110, and optionally expression of APLNR; or

[0123] - expression of the cell surface antigen CD110, and optionally expression of the cell surface antigen CD135; or

[0124] - expression of the cell surface antigen CD110, and optionally APLNR and CD135; or

[0125] - Expression of cell surface antigens CD135 and CD110 and expression of APLNR; or

[0126] - Expression of cell surface antigen CD135 and expression of APLNR; or

[0127] - Expression of cell surface antigen CD110 and expression of APLNR; or

[0128] - expression of APLNR, and optionally expression of the cell surface antigens CD135 and CD110; or

[0129] - expression of APLNR, and optionally expression of the cell surface antigen CD135; or

[0130] - Expression of APLNR, and optionally expression of the cell surface antigen CD110.

[0131] In embodiments where cells are sorted on the basis of expression of two or three markers, such as CD135, CD110, and APLNR, selection based on each of these markers can be performed simultaneously or sequentially in any order.

[0132] In a specific embodiment, in step b), cells are sorted on the basis of the expression of the cell surface antigen CD135 and / or CD110, preferably CD135 or CD110. In a preferred embodiment, in step b), cells are sorted on the basis of the expression of the cell surface antigen CD110 and optionally on the basis of the expression of the cell surface antigen CD135. In these embodiments, the method may further comprise sorting cells on the basis of the expression of APLNR before, after or simultaneously with step b).

[0133] In step c) of the method of the present invention, CD135+ and / or CD110+ and / or APLNR+ cells are recovered.

[0134] In a preferred embodiment, CD110+ cells are recovered.

[0135] When used in this article, the term "+" refers to the expression of the marker of interest preferably at the cell surface. For example, CD135+ cells are cells expressing the cell surface antigen CD135, and CD110+ / APLNR+ cells are cells expressing the cell surface antigens CD110 and APLNR. In contrast, the term "-" refers to the lack of expression of the marker of interest preferably at the cell surface. For example, CD135- cells are cells that do not express the cell surface antigen CD135, and CD110+ / APLNR- cells are cells that express the cell surface antigen CD110 and do not express APLNR.

[0136] Depending on the method for sorting cells, steps b) and c) may be sequential or simultaneous.

[0137] Depending on the marker used during the sorting step, the recovered cells can be CD135+ cells, CD110+ cells, APLNR+ cells, CD135+ / CD110+ cells, CD135+ / APLNR+ cells, CD110+ / APLNR+ cells or CD135+ / CD110+ / APLNR+ cells. Preferably, the cells are CD110+ cells, CD135+ / CD110+ cells, CD110+ / APLNR+ cells or CD135+ / CD110+ / APLNR+ cells.

[0138] These cells are capable of long-term multilineage engraftment and self-renewal and can be used for HSC transplantation.

[0139] If necessary, the method of the invention may comprise several sequential sorting steps based on the expression of CD135, CD110 and / or APLNR in order to enrich the cell product of CD135+, CD110+ and / or APLNR+ HSCs.

[0140] Optionally, prior to use, the cells may be stored, in particular may be cryopreserved, optionally in the presence of a cryoprotectant such as DMSO, for short or long term.

[0141] In another aspect, the present invention also relates to a method, preferably an in vitro method, for identifying and / or selecting hematopoietic stem cells suitable for hematopoietic transplantation, i.e. capable of long-term multi-lineage engraftment and self-renewal, comprising:

[0142] a) providing a cell population comprising hematopoietic stem cells, and

[0143] b) evaluating the expression of the cell surface antigens CD135 and / or CD110 and / or the expression of the apelin receptor (APLNR) of said cells, preferably the expression of the cell surface antigen CD110, and

[0144] c) identifying and / or selecting CD135+ and / or CD110+ and / or APLNR+ cells, preferably CD110+ cells.

[0145] All embodiments described above for the method of preparing a hematopoietic cell transplant of the present invention are also encompassed in this aspect.

[0146] The expression of the cell surface antigens CD135 and / or CD110 and / or the expression of the apelin receptor (APLNR) can be assessed by any method known to the skilled person, such as FACS, MACS, immunohistochemistry, Western blot, protein or antibody arrays, RT-PCR or by transcriptomic methods.

[0147] Depending on the method used to assess the expression of CD135, CD110 or APLNR, steps b) and c) may be sequential or simultaneous. For example, using FACS or MACS, the detection and selection of expression may be simultaneous.

[0148] The identified and / or selected CD135+ and / or CD110+ and / or APLNR+ cells can be used as a hematopoietic transplant, or can be included in or added to a hematopoietic transplant (eg, a bone marrow or umbilical cord blood transplant) in order to improve the efficacy of the transplant.

[0149] In another aspect, the present invention also relates to a method for producing transplantable HSCs from pluripotent stem cells, preferably an in vitro method, the method comprising:

[0150] Providing pluripotent stem cells, in particular iPSCs, preferably human iPSCs,

[0151] Induce embryoid body (EB) formation,

[0152] culturing the EBs in a liquid medium that induces differentiation of the pluripotent stem cells into endothelial-hematopoietic lineages,

[0153] Dissociate EB cells,

[0154] sorting the dissociated EB cells on the basis of the expression of the cell surface antigens CD135 and / or CD110 and / or apelin receptor (APLNR), preferably on the basis of the cell surface antigen CD110, and

[0155] CD135+, CD110+ and / or APLNR+ cells are recovered, preferably CD110+ cells.

[0156] All embodiments described above for the method of preparing a hematopoietic cell transplant of the present invention are also encompassed in this aspect.

[0157] As used herein, the term "transplantable HSC" refers to hematopoietic stem cells that are suitable for hematopoietic transplantation, ie, capable of long-term multi-lineage engraftment and self-renewal.

[0158] Preferably, the culture medium for inducing differentiation of pluripotent stem cells into endothelial-hematopoietic lineages comprises stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 (FLT3) ligand, bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1). Such culture medium may also comprise plasma, serum, platelet lysate, serum albumin, transferrin or its substitutes and / or insulin or its substitutes, preferably (i) plasma, serum and / or platelet lysate, and (ii) transferrin and insulin.

[0159] In a preferred embodiment, the culture medium for inducing differentiation of pluripotent stem cells toward the endothelial-hematopoietic lineage is a culture medium of the present invention and described hereinafter.

[0160] Preferably, the embryoid bodies are cultured in the liquid culture medium for 14 to 19 days, more preferably 15 to 18 days, even more preferably 17 days. In a preferred embodiment, the embryoid bodies are cultured in the liquid culture medium of the present invention and described hereinafter for 14 to 19 days, more preferably 15 to 18 days, even more preferably 17 days.

[0161] As demonstrated herein, CD135+, CD110+ and / or APLNR+ HSCs are long-term multipotent HSCs that support multi-lineage hematopoietic reconstitution and self-renewal in vivo and therefore constitute an excellent cell source for HSC transplantation.

[0162] Therefore, in another aspect, the present invention relates to the use of CD135, CD110 and / or APLNR as markers of hematopoietic stem cells suitable for hematopoietic transplantation, ie capable of engraftment, in particular long-term multi-lineage engraftment and self-renewal.

[0163] The present invention also relates to the use of CD135, CD110 and / or APLNR as a marker for assessing the efficacy of a hematopoietic cell transplant and / or as a marker for predicting the outcome and / or performance of a hematopoietic transplant.

[0164] The present invention also relates to a method for evaluating the efficacy of a hematopoietic cell transplant, preferably an in vitro method, comprising evaluating the presence or absence of HSCs expressing CD135, CD110 and / or APLNR in a hematopoietic cell transplant, preferably the presence or absence of HSCs expressing CD110, i.e., cells capable of long-term multi-lineage engraftment and self-renewal, the absence of which indicates low or absent efficacy. Conversely, the presence of HSCs expressing CD135, CD110 and / or APLNR can be considered to indicate good efficacy.

[0165] As used herein, the term "potency" refers to the specific ability of a cell product to affect a given outcome, and in particular refers to the ability of a hematopoietic cell product to provide in vivo multi-lineage hematopoietic reconstitution and self-renewal after transplantation, i.e., to regenerate the immune-hematopoietic system in a transplant patient.

[0166] Transplantation of a hematopoietic cell graft with low potency or deficiency may result in graft failure. Therefore, a hematopoietic cell graft that does not contain any HSC expressing CD135, CD110 and / or APLNR should not be used for transplantation.

[0167] The present invention also relates to a method for predicting the outcome of HSC transplantation, preferably an in vitro method, comprising detecting the presence or absence of HSC expressing CD135, CD110 and / or APLNR, preferably CD110, in a hematopoietic cell transplant, the absence of which indicates a poor prognosis, i.e. a high risk of graft failure. Conversely, the presence of HSC expressing CD135, CD110 and / or APLNR can be considered to indicate a good prognosis.

[0168] As used herein, the term "poor prognosis" refers to a decreased patient survival rate and / or a high risk of graft failure, i.e., a high risk that the graft cannot regenerate the immune-hematopoietic system in the transplanted patient. In contrast, the term "good prognosis" refers to an increased probability of improved patient survival and a successful transplant, i.e., an increased likelihood that the transplant allows regeneration of the immune-hematopoietic system in the transplanted patient.

[0169] The present invention also relates to a method for predicting the engraftment potential of a hematopoietic cell transplant, preferably an in vitro method, the method comprising detecting the presence or absence of HSC expressing CD135, CD110 and / or APLNR, preferably CD110, in a hematopoietic cell transplant, the absence of which indicates poor engraftment potential, i.e. a high risk of graft failure. Conversely, the presence of HSC expressing CD135, CD110 and / or APLNR can be considered to indicate good engraftment potential, i.e. an increased likelihood of successful transplantation.

[0170] The presence or absence of HSCs expressing CD135, CD110 and / or APLNR can be assessed by any method known to the skilled artisan or described above. For example, CD135+, CD110+ and / or APLNR+ cells can be detected using fluorescence activated cell sorting (FACS), magnetic activated cell sorting (MACS), or any immunoassay using antibodies to CD135, CD110 or APLNR. Monoclonal antibodies to CD135, CD110 or APLNR are commercially available.

[0171] The methods for assessing the efficacy of a hematopoietic cell transplant, predicting the outcome of an HSC transplant, or predicting the engraftment potential of a hematopoietic cell transplant as described above may also include any other phenotyping or functional assays routinely used by skilled artisans, such as counting the total number of viable nucleated cells (TNC), and / or measuring the number of functional progenitor cells capable of generating colonies of hematopoietic cells in a methylcellulose-based medium supplemented with stimulatory growth factors (CFU assay), and / or measuring the frequency of LTC-ICs.

[0172] In another aspect, the invention relates to a hematopoietic cell transplant prepared by any of the methods of the invention.

[0173] The present invention also relates to a hematopoietic cell transplant, wherein at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60% or 70% of the cells are CD135+, CD110+ and / or APLNR+ hematopoietic stem cells, preferably CD110+ hematopoietic stem cells, and further comprising a pharmaceutically acceptable carrier. Preferably, at least 70%, 75%, 80%, 85%, 90%, 95% or 99% of the cells of the hematopoietic cell transplant are CD135+, CD110+ and / or APLNR+ hematopoietic stem cells, preferably CD110+ hematopoietic stem cells. More preferably, at least 70%, 75%, 80%, 85%, 90%, 95% or 99% of the cells of the hematopoietic cell transplant are CD135+ and / or CD110+ HSC, preferably CD110+ HSC.

[0174] The proportion of CD135+, CD110+ and / or APLNR+ HSCs can be readily determined using any method known to the skilled artisan or described herein.

[0175] As used herein, the term "pharmaceutically acceptable" means approved for use in animals and / or humans by a regulatory agency or a recognized pharmacopoeia, such as the European Pharmacopoeia. The term "carrier" or "excipient" refers to a diluent, adjuvant, vehicle or medium with which the cell is administered. As is well known in the art, a pharmaceutically acceptable excipient is a relatively inert substance known to those skilled in the art, preferably an injectable substance.

[0176] All embodiments described above for the method of preparing a hematopoietic cell transplant of the present invention are also encompassed in this aspect.

[0177] In another aspect, the present invention relates to the use of the hematopoietic cell transplant of the present invention for treating various disorders associated with hematopoietic defects caused by diseases, disorders or myeloablative treatments, in particular for treating malignant or non-malignant diseases.

[0178] The present invention also relates to the use of the hematopoietic cell transplant of the present invention for the preparation of a medicament for the treatment of disorders associated with hematopoietic defects caused by a disease, disorder or myeloablative therapy, in particular for the treatment of malignant or non-malignant diseases.

[0179] The present invention also relates to a method for treating a disorder associated with hematopoietic deficiency caused by a disease, condition or myeloablative therapy, in particular for treating a malignant or non-malignant disease, in particular for treating a malignant or non-malignant disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a hematopoietic cell transplant of the present invention.

[0180] The present invention also relates to a method for treating a disorder associated with hematopoietic deficiency caused by a disease, disorder or myeloablative therapy, in a subject in need thereof, in particular for treating a malignant or non-malignant disease, said method comprising evaluating the efficacy of a hematopoietic cell transplant according to the method of the present invention as described above, and if said efficacy is good, administering to said subject an effective amount of said hematopoietic cell transplant.

[0181] All embodiments described above for the method of preparing a hematopoietic cell transplant, evaluating the efficacy of a hematopoietic cell transplant, or the hematopoietic cell transplant of the present invention are also encompassed in this aspect.

[0182] Examples of malignant diseases include, but are not limited to, multiple myeloma, non-Hodgkin's lymphoma, Hodgkin's disease, acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, myelodysplastic syndrome, myeloproliferative disorder, chronic lymphocytic leukemia, juvenile chronic myeloid leukemia, neuroblastoma, ovarian cancer, and germ cell tumors.

[0183] Examples of non-malignant diseases include, but are not limited to, autoimmune disorders, amyloidosis, aplastic anemia, paroxysmal nocturnal hemoglobinuria, Fanconi's anemia, Budd-Diesel anemia, thalassemia major, sickle cell anemia, severe combined immunodeficiency, Wieder-Oergler syndrome, and inborn errors of metabolism.

[0184] The term "subject" or "patient" refers to an animal, preferably a mammal, even more preferably a human, including an adult, a child, and a human in the fetal stage.

[0185] As used herein, the term "treatment" refers to any action intended to improve the patient's health status, such as treatment, prevention, prevention and delay of a disease. In certain embodiments, the term refers to improving or eradicating a disease or a symptom associated with a disease. In other embodiments, the term refers to minimizing the spread or worsening of the disease caused by the administration of one or more therapeutic agents to an object suffering from the disease. In certain embodiments, the term may refer to the regeneration of the immune-hematopoietic system in a transplant patient.

[0186] "Therapeutically effective amount" means the amount of hematopoietic cell transplant administered to a subject sufficient to constitute a treatment of a malignant or non-malignant disease as defined above. In certain embodiments, the term may refer to the amount of hematopoietic cell transplant necessary to regenerate the immune-hematopoietic system in a transplant patient.

[0187] The therapeutically effective amount may vary with the proportion of CD135+, CD110+ and / or APLNR+ cells in the hematopoietic cell transplant, with the patient's physiological data (eg age, size and weight) and the disease to be treated.

[0188] In one embodiment, administration of 10 4 Up to 10 7 , preferably 10 5 Up to 10 7 , more preferably 3.10 5 Until 6.10 6 CD135+, CD110+ and / or APLNR+ cells / kg patient body weight. In a specific embodiment, 10 5 Up to 10 6 , preferably 1×10 5 Up to 5×10 5 In another specific embodiment, 10 6 Up to 10 7 , preferably 3×10 6 Up to 8×10 6 APLNR+ cells / kg patient body weight.

[0189] The hematopoietic cell transplant according to the present invention can be used in combination with other therapies such as other chemotherapy, immunotherapy, radiotherapy or surgery, depending on the disease to be treated.

[0190] The term "immunotherapy" refers to therapeutic therapy that stimulates the patient's immune system to attack the malignant cells or cells that cause the disease. It includes immunizing the patient with a specific antigen (e.g., by administering a cancer vaccine), administering molecules that stimulate the immune system, such as cytokines, or administering therapeutic antibodies as drugs.

[0191] The term "radiotherapy" is a commonly used term in the art to refer to various types of radiation therapy, including internal and external radiation therapy, radioimmunotherapy, and to the use of various different types of radiation, including X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiation. Specifically, radiation therapy may be used to treat disease that may have spread outside the bone marrow to relieve bone pain, or for whole body irradiation prior to stem cell transplantation.

[0192] The chemotherapy can be used to treat malignant diseases and can include, for example, vincristine, daunorubicin, doxorubicin, idarubicin, mitoxantrone, cytarabine, asparaginase, etoposide, teniposide, mercaptopurine, methotrexate, cyclophosphamide, prednisone, dexamethasone, busulfan, hydroxyurea or interferon alpha, or any other relevant chemotherapy.

[0193] The hematopoietic cell transplant can be used for autologous, isogenic or allogenic transplantation. As used herein, "allogeneic transplantation" refers to the transplantation of cells derived from or originated from a donor that is genetically inconsistent with the recipient but belongs to the same species. "Autologous transplantation" refers to the transplantation of cells derived from or originated from the same subject. The donor and recipient are the same person. "Isogeneic transplantation" refers to the transplantation of cells derived from or originated from a donor that is genetically consistent with the recipient.

[0194] In a specific embodiment, the hematopoietic cell transplant is intended for autologous transplantation and the HSCs, in particular CD135+, CD110 and / or APLNR+ cells, are derived from induced pluripotent stem cells originating from the subject to be treated.

[0195] In another specific embodiment, the hematopoietic cell transplant is intended for allogeneic transplantation and the HSCs, particularly CD135+, CD110 and / or APLNR+ cells, are derived from placental blood or umbilical cord blood.

[0196] As shown in the experimental section, the inventors have developed a liquid cell culture medium in which the differentiation process of embryoid bodies obtained from pluripotent stem cells to the endothelial-hematopoietic lineage is delayed. Therefore, this culture medium allows the production and selection of early primitive hematopoietic stem cells, i.e., CD135+, CD110+ and / or APLNR+ HSCs, capable of long-term multi-lineage engraftment and self-renewal in vivo under GMP grade culture conditions.

[0197] Therefore, in another aspect, the present invention also relates to a liquid cell culture medium comprising or essentially consisting of the following components: (i) plasma, serum, platelet lysate and / or serum albumin, and (ii) transferrin or a substitute thereof, insulin or a substitute thereof, stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1). Preferably, the liquid cell culture medium comprises or consists essentially of the following components: (i) plasma, serum and / or platelet lysate, and (ii) transferrin, insulin, stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1).

[0198] As used herein, the term "cell culture medium" refers to any culture medium, in particular any liquid culture medium, comprising a basal medium that easily maintains the growth of eukaryotic cells, in particular mammalian cells, more particularly human cells. Basal culture media are well known to those skilled in the art.

[0199] As used herein, the term "consisting essentially of" refers to a culture medium that comprises (i) plasma, serum, platelet lysate and / or serum albumin, preferably plasma, serum and / or platelet lysate, and (ii) transferrin or a substitute thereof (preferably transferrin), insulin or a substitute thereof (preferably insulin), stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1), and does not comprise any other cytokine or growth factor.

[0200] In a specific embodiment, the culture medium of the present invention comprises Iscove's modified Dulbecco's medium (IMDM) optionally supplemented with glutamine or a glutamine-containing peptide as a basal medium, and to which are added (i) plasma, serum, platelet lysate and / or serum albumin, preferably plasma, serum and / or platelet lysate, and (ii) transferrin or a substitute thereof (preferably transferrin), insulin or a substitute thereof (preferably insulin), stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1).

[0201] Preferably, the culture medium of the present invention comprises 5 µg / mL to 20 µg / mL of insulin, more preferably 8 µg / mL to 12 µg / mL, even more preferably about 10 µg / mL of insulin. In a preferred embodiment, the insulin is human insulin, preferably human recombinant insulin.

[0202] Insulin substitutes can be any compound known to the skilled person that performs the same function as insulin in cell culture medium. Specifically, such substitutes can be any insulin receptor agonist, such as a small molecule or an aptamer agonist. Small molecule insulin receptor agonists have been described, for example, in Qiang et al., Diabetes. 2014 Apr; 63(4): 1394-409, and aptamer agonists have been described, for example, in Yunn et al., Nucleic Acids Res. 2015 Sep 18; 43(16): 7688-701. Preferably, insulin is replaced with a zinc salt, as described in Wong et al., Cytotechnology. 2004 Jul; 45(3): 107-15. Examples of zinc salts include, but are not limited to, zinc chloride, zinc nitrate, zinc bromide or zinc sulfate. In a preferred embodiment, the insulin substitute is a zinc salt. The concentration of the insulin substitute depends on the nature of the compound and can be easily determined by the skilled person.

[0203] Preferably, the culture medium of the present invention comprises 10 µg / mL to 100 µg / mL transferrin, preferably 30 µg / mL to 60 µg / mL transferrin, even more preferably about 45 µg / mL transferrin. In a preferred embodiment, the transferrin is iron-saturated human transferrin, preferably recombinant iron-saturated human transferrin.

[0204] Transferrin substitute can be any compound that performs the same function as transferrin in cell culture medium known to professionals. Specifically, transferrin can be replaced with iron chelators or inorganic iron salts such as ferric citrate, ferric nitrate or ferrous sulfate. Examples of suitable iron chelators include but are not limited to ethylenediaminetetraacetic acid (EDTA), edetic acid (EGTA), deferoxamine mesylate, dimercaptopropanol or pentetic acid (DPTA). The concentration of the transferrin substitute depends on the nature of the compound and can be easily determined by professionals.

[0205] The culture medium may comprise plasma, serum, platelet lysate and / or serum albumin, preferably plasma, serum and / or platelet lysate, more preferably plasma or serum or platelet lysate or serum albumin, even more preferably plasma or serum or platelet lysate. The culture medium may comprise 1% to 20% plasma or serum, preferably 2% to 10% plasma or serum, more preferably about 5% plasma or serum. In a preferred embodiment, the plasma or serum is human plasma or serum. Alternatively or in addition, the culture medium may comprise 0.1% to 2% platelet lysate, preferably 0.2% to 1% platelet lysate, more preferably about 0.5% platelet lysate. In a preferred embodiment, the platelet lysate is human platelet lysate. Alternatively or in addition, the culture medium may comprise 0.1% to 2% serum albumin, preferably 0.5% to 1% serum albumin. In a preferred embodiment, the serum albumin is human serum albumin.

[0206] Preferably, the culture medium of the invention comprises 10 ng / mL to 100 ng / mL of SCF, more preferably 10 ng / mL to 50 ng / mL of SCF, even more preferably about 24 ng / mL of SCF. In a preferred embodiment, the SCF is human SCF, preferably recombinant human SCF.

[0207] Preferably, the culture medium of the invention comprises 10 ng / mL to 100 ng / mL TPO, more preferably 10 ng / mL to 50 ng / mL TPO, even more preferably about 21 ng / mL TPO. In a preferred embodiment, TPO is human TPO, preferably recombinant human TPO.

[0208] Preferably, the culture medium of the present invention comprises 10 ng / mL to 100 ng / mL of FLT3-L, more preferably 10 ng / mL to 50 ng / mL of FLT3-L, even more preferably about 21 ng / mL of FLT3-L. In a preferred embodiment, FLT3-L is human FLT3-L, preferably recombinant human FLT3-L.

[0209] Preferably, the culture medium of the invention comprises 50 ng / mL to 300 ng / mL of BMP4, more preferably 150 ng / mL to 250 ng / mL of BMP4, even more preferably about 194 ng / mL of BMP4. In a preferred embodiment, the BMP4 is human BMP4, preferably recombinant human BMP4.

[0210] Preferably, the culture medium of the present invention comprises 50 ng / mL to 300 ng / mL VEGF, more preferably 150 ng / mL to 250 ng / mL VEGF, even more preferably about 200 ng / mL VEGF. In a preferred embodiment, VEGF is human VEGF, preferably recombinant human VEGF, more preferably recombinant human VEGF-A165.

[0211] Preferably, the culture medium of the invention comprises 10 ng / mL to 100 ng / mL IL3, more preferably 20 ng / mL to 80 ng / mL IL3, even more preferably about 50 ng / mL IL3. In a preferred embodiment, the IL3 is human IL3, preferably recombinant human IL3.

[0212] Preferably, the culture medium of the invention comprises 10 ng / mL to 100 ng / mL of IL6, more preferably 20 ng / mL to 80 ng / mL of IL6, even more preferably about 50 ng / mL of IL6. In a preferred embodiment, IL6 is human IL6, preferably recombinant human IL6.

[0213] Preferably, the culture medium of the invention comprises 1 ng / mL to 20 ng / mL IL1, more preferably 1 ng / mL to 10 ng / mL IL1, even more preferably about 5 ng / mL IL1. In a preferred embodiment, IL1 is human IL1, preferably recombinant human IL1.

[0214] Preferably, the culture medium of the invention comprises 10 ng / mL to 200 ng / mL GCSF, more preferably 50 ng / mL to 150 ng / mL GCSF, even more preferably about 100 ng / mL GCSF. In a preferred embodiment, the GCSF is human GCSF, preferably recombinant human GCSF.

[0215] Preferably, the culture medium of the invention comprises 1 ng / mL to 10 ng / mL of IGF1, more preferably 1 ng / mL to 10 ng / mL of IGF1, even more preferably about 5 ng / mL of IGF1. In a preferred embodiment, the IGF1 is human IGF1, preferably recombinant human IGF1.

[0216] In a specific embodiment, the liquid cell culture medium of the present invention comprises:

[0217] - 1% to 20% plasma or serum, preferably 2% to 10% plasma or serum; or 0.1% to 2% platelet lysate, preferably 0.2% to 1% platelet lysate; or 0.1% to 2% serum albumin, preferably 0.5% to 1% serum albumin; and / or

[0218] - 5 µg / mL to 20 µg / mL of insulin or a substitute thereof, preferably insulin, preferably 8 µg / mL to 12 µg / mL of insulin or a substitute thereof, preferably insulin; and / or

[0219] - 10 µg / mL to 100 µg / mL of transferrin or a substitute thereof, preferably transferrin, preferably 30 µg / mL to 60 µg / mL of transferrin or a substitute thereof, preferably transferrin; and / or

[0220] - 10 ng / mL to 100 ng / mL of SCF, preferably 10 ng / mL to 50 ng / mL of SCF; and / or

[0221] - 10 ng / mL to 100 ng / mL of TPO, preferably 10 ng / mL to 50 ng / mL of TPO; and / or

[0222] - 10 ng / mL to 100 ng / mL of FLT3-L, preferably 10 ng / mL to 50 ng / mL of FLT3-L; and / or

[0223] - 100 ng / mL to 500 ng / mL of BMP4, preferably 150 ng / mL to 250 ng / mL of BMP4; and / or

[0224] - 50 ng / mL to 300 ng / mL of VEGF, preferably 150 ng / mL to 250 ng / mL of VEGF; and / or

[0225] - 10 ng / mL to 100 ng / mL of IL3, preferably 20 ng / mL to 80 ng / mL of IL3; and / or

[0226] - 10 ng / mL to 100 ng / mL of IL6, preferably 20 ng / mL to 80 ng / mL of IL6; and / or

[0227] - 1 ng / mL to 20 ng / mL of IL1, preferably 1 ng / mL to 10 ng / mL of IL1; and / or

[0228] - 10 ng / mL to 200 ng / mL of GCSF, preferably 50 ng / mL to 150 ng / mL of GCSF; and / or

[0229] - 1 ng / mL to 20 ng / mL of IGF1, preferably 1 ng / mL to 10 ng / mL of IGF1.

[0230] Preferably, the culture medium meets all of these characteristics.

[0231] In another specific embodiment, the liquid cell culture medium of the present invention comprises:

[0232] - 1% to 20% plasma or serum, preferably 2% to 10% plasma or serum; or 0.1% to 2% platelet lysate, preferably 0.2% to 1% platelet lysate; and / or

[0233] - 5 µg / mL to 20 µg / mL of insulin, preferably 8 µg / mL to 12 µg / mL of insulin; and

[0234] - 10 µg / mL to 100 µg / mL of transferrin, preferably 30 µg / mL to 60 µg / mL of transferrin; and / or

[0235] - 10 ng / mL to 100 ng / mL of SCF, preferably 10 ng / mL to 50 ng / mL of SCF; and / or

[0236] - 10 ng / mL to 100 ng / mL of TPO, preferably 10 ng / mL to 50 ng / mL of TPO; and / or

[0237] - 100 ng / mL to 500 ng / mL of FLT3-L, preferably 250 ng / mL to 350 ng / mL of FLT3-L; and / or

[0238] - 10 ng / mL to 100 ng / mL of BMP4, preferably 10 ng / mL to 50 ng / mL of BMP4; and / or

[0239] - 50 ng / mL to 300 ng / mL of VEGF, preferably 150 ng / mL to 250 ng / mL of VEGF; and / or

[0240] - 10 ng / mL to 100 ng / mL of IL3, preferably 20 ng / mL to 80 ng / mL of IL3; and / or

[0241] - 10 ng / mL to 100 ng / mL of IL6, preferably 20 ng / mL to 80 ng / mL of IL6; and / or

[0242] - 1 ng / mL to 20 ng / mL of IL1, preferably 1 ng / mL to 10 ng / mL of IL1; and / or

[0243] - 10 ng / mL to 200 ng / mL of GCSF, preferably 50 ng / mL to 150 ng / mL of GCSF; and / or

[0244] - 10 ng / mL to 150 ng / mL of IGF1, preferably 10 ng / mL to 100 ng / mL of IGF1.

[0245] Preferably, the culture medium meets all of these characteristics.

[0246] In another specific embodiment, the liquid cell culture medium of the present invention comprises:

[0247] - 1% to 20% plasma or serum, preferably 2% to 10% plasma or serum; or 0.1% to 2% platelet lysate, preferably 0.2% to 1% platelet lysate; and / or

[0248] - 5 µg / mL to 20 µg / mL of insulin, preferably 8 µg / mL to 12 µg / mL of insulin; and / or

[0249] - 10 µg / mL to 100 µg / mL of transferrin, preferably 30 µg / mL to 60 µg / mL of transferrin; and / or

[0250] - 10 ng / mL to 100 ng / mL of SCF, preferably 10 ng / mL to 50 ng / mL of SCF; and / or

[0251] - 10 ng / mL to 100 ng / mL of TPO, preferably 10 ng / mL to 50 ng / mL of TPO; and / or

[0252] - 10 ng / mL to 100 ng / mL of FLT3-L, preferably 10 ng / mL to 50 ng / mL of FLT3-L; and / or

[0253] - 100 ng / mL to 500 ng / mL of BMP4, preferably 150 ng / mL to 250 ng / mL of BMP4; and / or

[0254] - 50 ng / mL to 300 ng / mL of VEGF, preferably 150 ng / mL to 250 ng / mL of VEGF; and / or

[0255] - 10 ng / mL to 100 ng / mL of IL3, preferably 20 ng / mL to 80 ng / mL of IL3; and / or

[0256] - 10 ng / mL to 100 ng / mL of IL6, preferably 20 ng / mL to 80 ng / mL of IL6; and / or

[0257] - 1 ng / mL to 20 ng / mL of IL1, preferably 1 ng / mL to 10 ng / mL of IL1; and / or

[0258] - 10 ng / mL to 200 ng / mL of GCSF, preferably 50 ng / mL to 150 ng / mL of GCSF; and / or

[0259] - 1 ng / mL to 20 ng / mL of IGF1, preferably 1 ng / mL to 10 ng / mL of IGF1.

[0260] Preferably, the culture medium meets all of these characteristics.

[0261] In another specific embodiment, the liquid cell culture medium of the present invention comprises: (i) about 5% plasma or serum or about 0.5% platelet lysate, and (ii) about 10 µg / mL insulin, about 45 µg / mL transferrin, about 22 ng / mL SCF, about 20 ng / mL TPO, about 300 ng / mL FLT3-L, about 22 ng / mL BMP4, about 200 ng / mL VEGF, about 50 ng / mL IL3, about 50 ng / mL IL6, about 5 ng / mL IL1, about 100 ng / mL GCSF, and about 50 ng / mL IGF1.

[0262] In another specific embodiment, the liquid cell culture medium of the present invention comprises: (i) about 5% plasma or serum or about 0.5% platelet lysate, and (ii) about 10 µg / mL insulin, about 45 µg / mL transferrin, about 24 ng / mL SCF, about 21 ng / mL TPO, about 21 ng / mL FLT3-L, about 194 ng / mL BMP4, about 200 ng / mL VEGF, about 50 ng / mL IL3, about 50 ng / mL IL6, about 5 ng / mL IL1, about 100 ng / mL GCSF, and about 5 ng / mL IGF1.

[0263] In embodiments where the culture medium comprises plasma or serum, it may advantageously further comprise heparin, preferably 0.5 U / mL to 5 U / mL of heparin, more preferably 2 U / mL to 4 U / mL of heparin, even more preferably about 3 U / mL of heparin.

[0264] The present invention also relates to the use of the liquid cell culture medium of the invention for the growth and / or differentiation of cells of the hematopoietic lineage, for the differentiation of embryoid bodies, for the production of hematopoietic cell transplants, in particular in the absence of feeder cells.

[0265] As used herein, the term "growth" refers to the proliferation of cultured cells, and the term "differentiation" refers to the acquisition of cell characteristics that make the cells become cells of the hematopoietic lineage by cells cultured in a culture medium. As used herein, the term "cell of the hematopoietic lineage" refers to cells present in the blood of mammals, particularly humans.

[0266] The cell culture medium of the present invention is particularly useful for the growth and / or differentiation of pluripotent stem cells, such as iPSCs, embryoid bodies, and HSCs, including early primitive HSCs, such as CD135+, CD110+ and / or APLNR+ HSCs.

[0267] All patents, patent applications, provisional applications, and publications mentioned or cited herein are incorporated by reference in their entirety, including all figures, to the extent not inconsistent with the explicit teachings of this specification.

[0268] The following examples are offered for purposes of illustration and not limitation. Example

[0269] Example 1

[0270] Materials and methods

[0271] hiPSC Expansion

[0272] The studies were performed using three different hiPSC lines: FD136-25, reprogrammed with retroviral vectors and Thomson's combination (endogenous expression of Oct4, Sox2, Nanog and Lin28); Pci-1426 and Pci-1432 lines (Phenocell), reprogrammed with episomes (Sox2, Oct4, KLF, cMyc). hiPSCs were maintained in TESR2 medium (Stem Cell Technologies, Bergisch Gladbach, Germany) on CellStart (Invitrogen, Carlsbad, USA), and cells were passaged 1:6 every 5 days on freshly coated plates using standard mass passaging with TRYpleselect (Invitrogen).

[0273] EB differentiation

[0274] After 24 h, cells were transferred to differentiation medium containing 24 ng / mL SCF, 21 ng / mL TPO, 21 ng / mL FLT3L, 194 ng / mL BMP4, 200 ng / mL VEGF, 50 ng / mL IL3, 50 ng / mL IL6, 5 ng / mL IL1, 100 ng / mL GCSF, 5 ng / mL IGF1 (PeproTech, Neuilly-sur-Seine, France). The medium was changed every other day. EBs were dissociated on days 15, 16, and 17.

[0275] Colony assay

[0276] At the specified time, 1x10 5 Dissociated EB cells or 3x10 4 Cells from the BM of xenograft recipients were plated in 3 mL of complete methylcellulose medium in the presence of SCF, IL-3, EPO, and GM-CSF (PeproTech, Neuilly-sur-Seine, France). Since G-CSF also stimulates mouse progenitor cells, it was replaced with granulocyte-macrophage colony-stimulating factor (GM-CSF). Aliquots (1 mL) of the mixture were dispensed twice into a 30 mm dish and maintained in a humidified incubator for 14 days. Colony-forming cells (CFCs) were scored on day 14.

[0277] Long-term culture-initiating cell assay

[0278] Long-term culture initiating cell (LTC-IC) assays were performed as previously described (see, e.g., Miller and Eaves, Hematopoietic Stem Cell Protocols, Volume 63 of the series Methods in Molecular Medicine pp 123-141). 15-100,000 cells / well for EBs at day 17 and for control CD34+ at day 0. Absolute LTC-IC counts corresponded to cell concentrations, using Poisson statistics to give 37% negative wells.

[0279] Microtubule-like and EPC-like cells

[0280] For pseudo-microtubule formation, cells were transferred onto growth factor-reduced Matrigel (Corning) and cultured in EGM2 medium (Lonza).

[0281] For the generation of EPC-like cells, cells were first plated on gelatin and cultured in EBM2 (Lonza) and split several times, with gelatin no longer being mandatory after the first passage.

[0282] Flow cytometry

[0283] For staining of BM cells or dissociated EBs, 2x10 5 Each cell was incubated in 100 μL staining buffer (PBS containing 2% FBS) containing each antibody at a 5:100 dilution for 20 min at room temperature in the dark. Data acquisition was performed on a Becton Dickinson Canto II cytometer.

[0284] In vivo analysis of angiogenic potential

[0285] 1.750×10 6 D16 single cells or hEPC and 1.750×10 6 hMSCs were mixed with 100 μl of phenol red-free and growth factor-reduced Matrigel (Corning) and injected subcutaneously into the back of nude mice (two different plugs / mouse). Controls were performed similarly, but using 3.5 × 10 6hMSC or D16 single cells or hEPC; n = 3 for each condition. Two weeks later, mice were sacrificed, matrigel plugs were removed and processed for paraffin sections. Sections were deparaffinized, dehydrated and stained with either Masson's trichrome, a three-color scheme consisting of nuclear staining with hematoxylin, cytoplasmic staining with acid fuchsin / xykidine ponceau, and collagen staining with brilliant green SF (all from WR); or human von Willebrand factor (Dako), developed with histogreen substrate (Abcys) and counterstained with nuclear fast red (DakoCytomation), dehydrated and mounted; or hCD31 (R&D system) as primary antibody and donkey anti-rabbit Cy3 antibody (Jackson Immuno Research) and DAPI as secondary antibodies, and mounted with fluoromount G.

[0286] Sorting of APLNR-positive cells

[0287] Cells were stained with antibody hAPJ-APC as described above. Sorting was performed on a Moflo ASTRIOS Beckman Coulter apparatus and the purity was 98.1% APLNR positive cells.

[0288] Mouse transplantation

[0289] NOD / SCID-LtSz-scid / scid(NOD / SCID) or NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ (NSG) or Foxn1− / − nude mice (Charles River, L'Abresle, France) were housed in the IRSN animal care facility. All experiments and procedures were performed in accordance with the regulations of the French Ministry of Agriculture on animal experiments and were approved by the local ethics committee.

[0290] 24 h before cell injection, mice aged 6-8 weeks and housed under sterile conditions were sublethally irradiated with 2.5 Gy from a 137Cs source (2.115 Gy / min). To ensure consistency between experiments, only male mice were used. Before transplantation, mice were temporarily sedated by intraperitoneal injection of ketamine and xylazine. Cells (0.4 x10 6 A total of 140 mice were used in this study.

[0291] Regarding the engraftment potential of D17 cells from three different hiPSC lines:

[0292] 70 NSG mice were used as follows: 30 as primary recipients, 30 as secondary recipients, and 10 as controls.

[0293] 48 NOD-SCID mice were used as follows: 20 as primary recipients, 16 as secondary recipients, 3 as tertiary recipients, and 9 as controls.

[0294] For engraftment potential of APLNR+ and APLNR- populations: 10 NOD-SCID mice were used, and 3 NOD-SCID as controls.

[0295] In vivo assessment of endothelial and hematopoietic potential was explored in 9 nude mice.

[0296] Assessment of human cell engraftment

[0297] Mice were sacrificed at 12, 18, or 20 weeks. Femurs, tibiae, livers, spleens, and thymuses were removed. Single-cell suspensions were prepared by standard washes and 1 × 10 6 Aliquots of cells were stained in a total volume of 200 μL of staining buffer.

[0298] Samples were stained for engraftment assessment using the following markers: hCD45 clone J33, hCD43 clone DFT1, hCD34 clone 581 (Beckman Coulter) and hCD45 clone 5B1, mCD45 clone 30F11 (Miltenyi).

[0299] BM was pooled to allow hCD45 microbead enrichment (Miltenyi) and multilineage assessment was performed using the following human markers: hCD3 clone UCHT1, hCD4 clone 13B8.2, hCD8 clone B9.11, hCD14 clone RMO52, hCD15 clone 80H5, hCD19 clone J3-119, hCD20 clone B9E9, hCD41 clone P2, hCD61 clone SZ21, hCD43 clone DFT1, hCD34-APC, hCD71 clone YDJ1.2.2 (all from Beckman Coulter antibodies, Brea, USA), CD45 clone 5B1 (Miltenyi), CD235a clone GA-R2 (Becton-Dickinson).

[0300] Blood samples were pooled and allowed to undergo hCD45 microbead sorting (Miltenyi). Multi-lineage potential was assessed using the following human markers: hCD3 clone UCHT1, hCD4 clone 13B8.2, hCD8 clone B9.11, hCD14 clone RMO52, hCD15 clone 80H5, hCD19 clone J3-119, hCD20 clone B9E9, hCD41 clone P2, hCD61 clone SZ21 (all from Beckman Coulter antibodies, Brea, USA).

[0301] BM from uninjected mice was used as a control for nonspecific staining.

[0302] Data were acquired on a BD Canto II cytometer using the FMO method with compensation using anti-mouse Ig antibodies.

[0303] T-cell maturation and function assays

[0304] Blood from three mice was pooled to allow hCD2 microbead sorting (Miltenyi) and assessed for the presence of TCR αβ and TCR γδ by flow cytometry using the following human markers: TCR αβ clone IP26A and TCR γδ clone IMMU510 (both from Beckman Coulter antibodies, Brea, USA).

[0305] Thymic and splenocytes were isolated, labeled with CFSE, and plated in cell culture medium supplemented or not with hCD3 and hCD28 (Beckman Coulter, both at 1 μg / ml). After 5 days, cells were harvested, stained with anti-hCD3 antibody clone UCHT1, and analyzed on a BD Canto II cytometer. CD3 was analyzed using FlowJo analysis software. + T-cells were gated and overlay histograms were generated.

[0306] To assess the presence of thymocytes, thymocytes were labeled with hCD1A clone BL6 (from Beckman Coulterantibodies, Brea, USA).

[0307] Evaluation of the safety of APLNR cells

[0308] Three sublethally irradiated NOD / SCID mice were each injected subcutaneously with 3 million APLNR-positive cells. No teratomas were found after 2 months of follow-up in accordance with FDA guidelines (Materials and Methods).

[0309] Furthermore, no tumors were macroscopically detected in any of the mice after organ analysis (140 / 140 mice) or after microscopic analysis of different tissues (brain, lung, kidney, BM, liver, and intestine) (140 / 140 mice).

[0310] Quantitative PCR

[0311] Total mRNA was isolated using an RNA mini kit (Qiagen, Courtaboeuf, France). mRNA integrity was checked on a Bioanalyzer 2100 (Agilent Technologies, Massy, ​​France). cDNA was constructed by reverse transcription using Superscript (Life Technologies, Carlsbad, USA). PCR assays were performed using TaqMan PCR Master Mix (Life Technologies) and specific primers for selected genes (Applied BioSystems, Carlsbad, USA) (see table below) with a sequence detection system (QuantStudio™ 12K Flex Real-Time PCR System, Life Technologies). In each sample, the fluorescent PCR signal of each target gene was normalized to the fluorescent signal of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

[0312] The human origin of mRNA from mouse BM was assessed by measuring hCD45, hCD15, hMPO, hITGA2, and hGAPDH. From the expression of globin classes in transplanted CFCs and mouse BM, we measured β, γ, and ε globin using Taqman probes.

[0313] The control was CD34 + The resulting cultured erythroblasts.

[0314]

[0315] Statistical analysis

[0316] All statistics were determined using R software 3.1.1 (2014-07-10) (R Core Team, 2013), INGENUITY and SAM software. Data were presented using hierarchical clustering and PCA.

[0317] result

[0318] The first transplantable HSCs are generated during embryonic development from a specialized population of endothelial cells (ECs) termed hemogenic endothelium. Following the endothelial-to-hematopoietic transition (EHT), these hemogenic ECs differentiate into hematopoietic cells (HCs), including HSCs, enter the circulation, expand in the embryonic liver, and arrive at their final residence, the BM. These early steps of developmental hematopoiesis, especially the generation of hemogenic ECs and budding of HCs, are fully recapitulated in embryoid body (EB) cultures.

[0319] The inventors have developed a one-step, carrier-free and matrix-free systematic procedure to direct the differentiation of hiPSCs into the endothelial-hematopoietic lineage. From day 0 (D) to the end of the culture period, all cytokines and growth factors are present to meet any needs. Many studies use a 14-day long protocol and isolate cells between D11 and D14 based on the presence of a hematopoietic burst on EBs. The inventors did not obtain a burst even at D17, thus evaluating this as a dramatic delay in the differentiation process ( Figure 1 A). Applying these culture conditions to three different hiPSC lines with different reprogramming protocols, such as using episomes or retroviruses, resulted in similar differentiation potency, thus confirming the robustness of the approach.

[0320] To determine the time point of hematopoietic EC / early HC commitment, the present inventors analyzed the expression of pluripotent genes and 49 key endothelial and hematopoietic specific genes of EB cells by qRT-PCR at D3, D7, D9, D13, D15, D16 and D17, with CD34 + The molecular pattern of cord blood HSC was used as reference. Hierarchical cluster analysis ( Figure 1 B) shows that there are two major groups, one with CD34 + The latter were divided into two distinct clusters: one cluster contained early EB cells (D3 to D13) and the other encompassed late EB cells (D15 to D17), suggesting that there is a balance point between D13 and 15. A closer analysis of the qPCR patterns identified D13 as the time point of EC commitment based on the expression of CD309 (VEGFR2) mRNA and D16 as the time point of putative hemogenic endothelial commitment based on the expression of RUNX1 mRNA ( Figure 1 C). From D16 onwards, hematopoietic-specific markers such as ITGA2 (integrin alpha-2) and CEBPA (CCAAT enhancer binding protein alpha) were also upregulated, coinciding with the onset of RUNX1 expression. D17 cells showed a shift towards CD34 +There was a trend of cell pattern convergence, as indicated by the increased expression of HC-specific genes (data not shown). To confirm the equilibrium point, the inventors analyzed the surface expression of CD309 as an EC marker and MPL, CKIT, and ITGA2 as early HC markers of the cell population by flow cytometry. Flow cytometric analysis confirmed a decrease in CD309 from D13 to D17, and an increase in ITGA2, CKIT, and MPL ( Figure 1 D), which is consistent with q-PCR analysis ( Figure 1 They further identified a cell population that expressed the APELIN receptor (APLNR), which is associated with early hematopoietic commitment, on D15 to D17 cells, and within this, a sub-fraction that progressively acquired expression of the motility and homing receptor CXCR4 ( Figure 1 E).

[0321] They then evaluated the endothelial and hematopoietic potential of EB cells at D15, D16, and D17 using dedicated in vitro functional assays ( Figure 2 A). D15 cells exhibit strong endothelial-forming potential, as evidenced by their generation of colony-forming endothelial cells (CFC-EC) ( Figure 2 A1), pseudo-microtubules ( Figure 2 A2) and EC-like cells ( Figure 2 A3) but lacked hematopoietic capacity, could not generate clonogenic colonies, and exhibited a very low frequency of long-term culture-initiating cells. In contrast, D17 cells lacked endothelial potential but exhibited a significantly improved hematopoietic capacity ( Figure 2 A4, Figure 2 A5), confirming the onset of hematopoietic commitment during this period.

[0322] The D16 equilibrium point was probed in vivo by subcutaneously transplanting cells in Matrigel (growth factor-reduced) plugs with or without human mesenchymal stem cells (hMSCs) into immunocompromised Foxn1− / − (nude) mice ( Figure 2 B) Human CD31 was detected in grafts containing D16 cells and / or hMSCs 2 weeks after transplantation. + Von Willebrand Factor + Human blood vessel structures made of cells ( Figure 2 C. Figure 2D). QRT PCR revealed expression of hVEGFR2, hENG (ENDOGLIN), hPECAM-1 in grafts made from D16 cells / hMSCs and, as expected, in grafts made from EPCs / hMSCs (data not shown). In addition, D16 cell / hMSC plugs expressed human β, γ, and ε globin transcripts, whereas D16 cell plugs alone expressed only human ε globin transcripts, revealing a block in maturation. Thus, D16 cells exhibit a balanced endothelial-hematopoietic pattern, consistent with the in vitro results.

[0323] Since D17 cells showed the strongest hematopoietic ability, the inventors used 4×10 5 Cells were transplanted into 8-week-old immunocompromised mice that were sublethally irradiated (3.5 Gy) for 20 weeks and then challenged secondary transplanted into similarly treated immunocompromised recipients for an additional 20 weeks ( Figure 3 A). The presence of human HCs was quantified by their surface expression of hCD34, hCD43, and hCD45 ( Figure 3 B. Figure 3 C. Figure 3 D). Multi-lineage human hematopoiesis is evident in 30 / 30 primary recipient mice ( Figure 3 C) The mean hCD45 in total mouse BM mononuclear cells was 20.3 + / - 2.9%. + cells, which is 203 times the 0.1% threshold that is usually considered positive for engraftment of human HC in NSG mice ( ), and 12.2+ / -1.5% hCD43 + and 7.29+ / -1.0% hCD34 + ( Figure 3 C). In the hCD45 + In the BM population, several human HC lineages were detected, including B cells (CD19 + CD45 + ), T cells (CD3 + CD45 + 、CD4 + CD45 + ), macrophages (CD14 + CD45 + 、CD15 + CD45 + )( Figure 3 E, Figure S3H) and erythroid progenitor / precursor cells (CD235a + CD45 + ) (not shown). Sorted hCD45 +Peripheral blood cells showed the same multi-lineage pattern, indicating the peripheralization of the transplanted cells. The human origin of the engrafted cells was confirmed by q-PCR using human-specific primers for CD45, CD15, MPO, ITGA2, and GAPDH genes (n=30 / 30). Human-specific clonogenic assays performed on BM cells isolated from primary recipient mice revealed that the human 4 Total BM cells ( Figure 3 F) showed an overall frequency of 17.5 + / - 4.3 clones distributed among CFU-GEMM, BFU-E, and CFU-GM colonies ( Figure 3 G1, Figure 3 G2, Figure 3 G3). Cytospin analysis revealed the presence of mature macrophages, tissue monocytes, myeloid cells, and erythroblasts ( Figure 3 H1, Figure 3 H2, Figure 3 H3). 7×10 6 BM cells were challenged in secondary (n = 30) recipients ( Figure 3 B. Figure 3 D), and in the case of NOD-SCID mice, challenged in tertiary recipients (n = 3) (data not shown). Human CD45 + Cells accounted for 12.6+ / -3.9% of mononuclear BM cells ( Figure 3 B. Figure 3 D), indicating sustained reconstitution capacity. Multilineage engraftment was found in 30 / 30 mice ( Figure 3 E). In 10 4 The overall cloning efficiency of human CFCs in total mouse BM cells was 5.5 + / - 3.1%, indicating a robust and sustained self-renewal capacity ( Figure 3 FH). The human origin of the implanted cells was confirmed as described above.

[0324] To ensure the functionality of the transplanted cells, the inventors analyzed the ability of human erythroid progenitor cells from mouse bone marrow to undergo hemoglobin switching in vivo and tested the phenotype and function of T cells. Implanted cells from both primary and secondary recipients were able to generate human erythroid progenitor cells that exhibited large amounts of β (39.51+ / -4.95 and 36.61+ / -5.86, respectively) and γ globin (57.49+ / -3.95 and 61.39+ / -4.86, respectively), while ε globin was dramatically reduced to 3.0+ / -1.2% and 2.1+ / -1.1% of total globin, respectively ( Figure 3 I). Silencing of embryonic globin expression and activation of adult globin expression are hallmarks of definitive erythroid cells. hCD2 isolated from peripheral blood +T cells express a large number of TCRαβ ( Figure 3 J) and very small amounts of TCRγδ to assess the maturation capacity of human T cells. The in vitro expansion capacity of thymic and spleen cells was tested after stimulation with hCD3 and hCD28, as measured by CSFE labeling. After 5 days, hCD3 + Thymus expressing gating ( Figure 3 K) and spleen (data not shown) cells showed tremendous expansion capacity, confirming the functionality of human T cells.

[0325] Figure 4 A shows APLNR in EBs at the early stage of culture. + The percentage of hCD45 cells in primary NOD-SCID recipients 18 weeks after transplantation was + The present inventors sorted APLNR + and APLNR - The APLNR groups were selected and their engraftment abilities were compared in the NOD-SCID model. + The cells successfully reconstituted hematopoiesis after 18 weeks ( Figure 4 B). In 6 / 6 transplanted mice, human CD45 + The cells accounted for 6.6+ / -1.9% of the mononuclear cells in the mouse BM, and 3.4+ / -2.5% were hCD43 + and 1.1+ / -0.4% were hCD34 + ( Figure 4 B. Figure 5 Flow cytometric analysis of BM cells revealed a human multi-lineage phenotype (data not shown). D17 APLNR + Cells do not contain any CD45 + cells, thus indicating that the reconstitution capacity is not mediated by hCD45 + The presence of committed progenitor cells results in Figure 4 C). In contrast, in 4 / 4 mice, APLNR - Cells failed to engraft at significant levels, with only 0.08 + / - 0.01% hCD45 + cell( Figure 4 B and Figure 5 ). Interestingly, the APLNR + The fractions exhibited ENG as described in mice + / TIE + / CKIT + A uniform group ( Figure 4 C) to enhance definitive hematopoiesis.

[0326] To further characterize the APNLR +The inventors of this invention will APLNR + and APLNR - Molecular patterns of cells compared with hiPSC and control CD34 + The molecular patterns of HSCs were compared in terms of the expression of gene sets representing the pluripotent state and endothelial, hemogenic endothelial, or hematopoietic commitment. Using the panel of 49 mRNAs studied as variables and the six cell populations as observations, principal component analysis (PCA) of gene expression levels represented by ∆Ct ( Figure 4 D) revealed that the first component, which may correspond to the factor “hematopoietic differentiation”, accounted for 44.9% of the variance. To further reveal traits involved in transplantation potential, they used PCA to separate APLNR + , D17 and HSC populations and APLNR - The third component, accounting for 19.23% of the variance, divided the population into two groups with different transplantation potential ( Figure 4 E). The statistical SAM test, which measures the strength of the relationship between gene expression and the response variable, indicated eight genes that were significantly more upregulated in the non-transplantable group (FDR < 10%), among which the endothelial genes were TEK, PECAM, and KDR ( Figure 4 F).

[0327] On the basis of these findings, the inventors showed the generation of long-term pluripotent HSCs that support multi-lineage hematopoietic reconstitution and in vivo self-renewal, through early differentiated cells that undergo EHT and express APLNR. These experiments were performed under GMP-grade culture conditions, thus paving the way for the use of pluripotent stem cells as a preferential source of cells for HSC transplantation.

[0328] Example 2

[0329] Materials and methods

[0330] hiPSC expansion, EB differentiation, assessment of human cell engraftment, T cell maturation and functional assays, quantitative PCR were performed as described above.

[0331] Cell sorting

[0332] Dissociated EB cells were stained with antibodies CD110-PE (MPL) or CD135-PE (FLT3), then stained again with PE-MicroBeads (Miltenyi), and finally sorted using a MACS® cell separation device.

[0333] Mouse transplantation

[0334] NOD.Cg-Prkdc scid Iq tm1Wjl / SzJ(NSG) (Charles River, L'Abresle, France) were housed in the IRSN animal care facility. All experiments and procedures were performed in accordance with the regulations of the French Ministry of Agriculture on animal experiments and were approved by the local ethics committee.

[0335] 24 h before cell injection, mice aged 6-8 weeks and housed under sterile conditions were sublethally irradiated with 3.5 Gy from a 137Cs source (2.115 Gy / min). To ensure consistency between experiments, only male mice were used. Before transplantation, mice were temporarily sedated by intraperitoneal injection of ketamine and xylazine. MPL+ or FLT3+ cells (10 4 / mouse) transplantation.

[0336] Regarding the engraftment potential of D17 cells from three different hiPSC lines:

[0337] Fifty NSG mice were used as follows: 25 as primary recipients and 25 as secondary recipients.

[0338] Bioinformatics

[0339] Bioinformatics analysis was performed in R environment software version 3.0.2. Publicly available transcriptome datasets were downloaded as normalized matrices (GSE format: gene expression matrix) from the database Gene Expression Omnibus (GEO) (http: / / www.ncbi.nlm.nih.gov / geo / ).

[0340] result

[0341] In order to better characterize the differentiated scid regenerative cells (SRC) from pluripotent cells (IPSC: induced pluripotent cells), the inventors analyzed the transcriptome samples, considering their xenotransplantation ability for successful primary or secondary transplantation. The transcriptome series were combined to establish a control group of sorted hematopoietic stem cells (HSC, with phenotype: CD34+CD38-CD90+, n=3) to compare with a group of IPSCs with only primary xenotransplantation ability (GI group, n=3) and a group of IPSCs with primary and secondary xenotransplantation ability (GI&GII group, n=3). After mathematical correction for batch effects, a one-way ANOVA (analysis of variance, p-value less than 1E-4) supervised analysis was performed between the 3 defined sample groups (HSC, GI, GI&II). Unsupervised principal component analysis of 5859 differential genes between the groups allowed significant differentiation of the sample groups with a p-value of 4.75E-8 on the main plot ( Figure 6). These results suggest that the selected genes may be suitable for studying the xenograft phenotype of SRC-IPSCs taking into account their ability to provide primary and / or secondary engraftment. Furthermore, batch effects associated with the transcriptomic dataset showed no impact on phenotypic group distinction during this unsupervised analysis. Supervised analysis was performed by significance analysis of microarrays (SAM) between each xenograft group and the HSC group in order to discover HSC biomarkers in each SRC-IPSC group. In the relevant Circos plots ( Figure 7 ), a higher diversity of HSC biomarkers was found in the GI&GII group compared to the GI group. This specific diversity of the GI&GII group included multiple functional categories, such as: mesoderm, pluripotent stem cells, and IPSCs. The specificity of the biomarkers in the GI group involved more mesenchymal phenotypes such as osteoblasts and adipocytes. On the other hand, common biomarkers were more present in the hematopoietic lineage, such as hematopoietic progenitors, erythroblast progenitors, CD34+ cells, bone marrow, and CD14+ cells. In order to observe the impact of HSC expression (CD34+38-90+) in the characterization of SRC-IPSCs, the HSC group was introduced in the supervised analysis to compare the SRC-IPSC group. The expression heat map performed by unsupervised classification showed that each xenograft SRC-IPSC group expressed certain HSC-related biomarkers: HSC biomarkers associated with the GI group of SRC-IPSCs, and HSC biomarkers associated with the GI&GII group of SRC-IPSCs (data not shown). A Venn diagram comparing HSC biomarkers enriched in each SRC-IPSC group shows any common genes ( Figure 8 ). SRC-IPSC cells with GI&GII competence specifically expressed certain cell surface molecules compared with the GI group: FLT3 (CD135) and MPL (CD110).

[0342] On the basis of this computer simulation, the inventors decided to study the MPL and FLT3 receptors more specifically, since antibodies are available that allow immunomagnetic screening for them.

[0343] After 17 days of hiPSC differentiation in EBs in a suitable medium (see Example 1), the inventors performed screening and then transplanted 10.000 cells / NSG immunosuppressed mice (n=15 for FLT3 and n=15 for MPL) ( Fig. 9 After 20 weeks, the mice were sacrificed and their bone marrow, spleen, liver and thymus, as well as blood samples, were studied.

[0344] For both populations (FLT3+ cells and MPL+ cells), high levels of engraftment were obtained (12.6+ / - 0.7% hCD45+ for the FLT3+ population and 9.9+ / - 1.7% for the MPL+ population) ( Fig.10 ), and human cells from all hematopoietic lineages were found. It was found that human erythrocytes produce β-globin, circulating T lymphocytes expressing TCRγδ on their surface, and lymphocytes from the thymus or spleen were able to proliferate in vitro after activation, suggesting that FLT3+ or MPL+ cells are capable of definitive hematopoiesis.

[0345] For each primary mouse, 7 million bone marrow cells were transplanted into secondary mice. After 20 weeks, these secondary mice were sacrificed and analyzed as described above.

[0346] All secondary mice showed high levels of engraftment (15.2+ / -3.4% hCD45+ for FLT3+ population, 9.8+ / -2.1% for MPL+ population) ( Fig.10 ), confirmed multi-lineage, and the transplanted human cells were capable of definitive hematopoiesis.

[0347] Therefore, cells obtained by differentiation of hiPSCs according to the inventors' protocol and expressing FLT3 or MPL on their surface are capable of long-term multi-lineage engraftment and self-renewal in vivo.

Claims

1. A liquid cell culture medium comprising (i) plasma, serum, platelet lysate and / or serum albumin, and (ii) transferrin or a substitute thereof, preferably transferrin, insulin or a substitute thereof, preferably insulin, stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1).

2. The liquid cell culture medium according to claim 1, comprising (i) plasma, serum and / or platelet lysate, and (ii) transferrin, insulin, stem cell factor (SCF), thrombopoietin (TPO), FMS-like tyrosine kinase 3 ligand (FLT3-L), bone morphogenetic protein 4 (BMP4), vascular endothelial growth factor (VEGF), interleukin 3 (IL3), interleukin 6 (IL6), interleukin 1 (IL1), granulocyte colony stimulating factor (GCSF) and insulin-like growth factor 1 (IGF1).

3. The liquid cell culture medium according to claim 1 or 2, comprising - 10 to 100 ng / mL SCF; and / or - 10 to 100 ng / mL TPO; and / or - 10 to 100 ng / mL of FLT3-L; and / or - 50 to 300 ng / mL of BMP4; and / or - 50 to 300 ng / mL of VEGF; and / or - 10 to 100 ng / mL IL3; and / or - 10 to 100 ng / mL IL6; and / or - 1 to 20 ng / mL IL1; and / or - 10 to 200 ng / mL GCSF; and / or - 1 to 20 ng / mL of IGF1.

4. The liquid cell culture medium according to claim 1 or 2, comprising - SCF between 10 and 50 ng / mL; and / or - 10 to 50 ng / mL TPO; and / or - 10 to 50 ng / mL of FLT3-L; and / or - 150 to 250 ng / mL of BMP4; and / or - 150 to 250 ng / mL VEGF; and / or - 20 to 80 ng / mL IL3; and / or - 20 to 80 ng / mL IL6; and / or - 1 to 10 ng / mL IL1; and / or - GCSF 50 to 150 ng / mL; and / or - 1 to 10 ng / mL of IGF1.

5. The liquid cell culture medium according to any one of claims 1 to 4, comprising 1% to 20% plasma or serum, preferably 2% to 10% plasma or serum. 6 . The liquid cell culture medium according to claim 1 , comprising 0.1% to 2% platelet lysate, preferably 0.2% to 1% platelet lysate.

7. The liquid cell culture medium according to any one of claims 1 to 6, comprising 0.1% to 2% serum albumin, preferably 0.5% to 1% serum albumin.

8. The liquid cell culture medium according to any one of claims 1 to 7, comprising 5 to 20 µg / mL of insulin or a substitute thereof, preferably insulin, preferably 8 to 12 µg / mL of insulin or a substitute thereof, preferably insulin.

9. The liquid cell culture medium according to any one of claims 1 to 8, comprising 10 µg / mL to 100 µg / mL of transferrin or a substitute thereof, preferably transferrin, preferably 30 µg / mL to 60 µg / mL of transferrin or a substitute thereof, preferably transferrin.

10. The liquid cell culture medium according to any one of claims 1 to 9, comprising plasma or serum, and further comprising heparin, preferably 0.5 U / mL to 5 U / mL of heparin.

11. Use of the liquid cell culture medium according to any one of claims 1 to 10 for the growth and / or differentiation of cells of the hematopoietic lineage, for the differentiation of embryoid bodies, and / or for the production of hematopoietic cell transplants.

12. The use according to claim 11, wherein the liquid cell culture medium is used in the absence of feeder cells.

13. An in vitro method for preparing a hematopoietic cell transplant or enriching hematopoietic stem cells capable of long-term multi-lineage engraftment and self-renewal from a cell population, the method comprising: a) providing a cell population comprising hematopoietic stem cells, and b) sorting cells of said population on the basis of the expression of cell surface antigens CD135 and / or CD110, and c) recovering CD135+ and / or CD110+ cells, And the method further comprises before step a): Provide pluripotent stem cells, Induce embryoid body (EB) formation, culturing the EBs in a liquid cell culture medium according to any one of claims 1 to 9, and Dissociate EB cells, The cell population provided in step a) is thus obtained.

14. The method according to claim 13, further comprising, before, after or simultaneously with step b), sorting cells based on the expression of apelin receptor (APLNR) and recovering APLNR+ cells.

15. The method according to any one of claims 13 and 14, wherein the pluripotent stem cells are selected from induced pluripotent stem cells or embryonic stem cells, preferably induced pluripotent stem cells. 16 . The method according to claim 13 , wherein the pluripotent stem cells are cultured in the liquid culture medium for 14 to 19 days, preferably 15 to 18 days, more preferably 17 days.

Images