WO2018167317A1 - Procédé d'expansion in vitro de cellules souches - Google Patents

Procédé d'expansion in vitro de cellules souches Download PDF

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WO2018167317A1
WO2018167317A1 PCT/EP2018/056766 EP2018056766W WO2018167317A1 WO 2018167317 A1 WO2018167317 A1 WO 2018167317A1 EP 2018056766 W EP2018056766 W EP 2018056766W WO 2018167317 A1 WO2018167317 A1 WO 2018167317A1
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coa
malonyl
malonic acid
stem cell
hematopoietic stem
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Sebastian Jessberger
Marlen Knobloch
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Universität Zürich
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation

Definitions

  • the present invention relates to a method of stem cell expansion, and to the use of malonate, malonyl-CoA and derivatives or conjugates thereof in the treatment of neuropsychiatric disorders and conditions requiring hematopoietic stem cell transplantation.
  • Hematopoietic stem cell transplantation is used in the therapy of many conditions including hematological malignancies, solid tumors, immunodeficiencies and hematologic diseases like anemias.
  • HSCs hematopoietic stem cells
  • the HSCs are then administered back to the patient to replace the destroyed bone marrow.
  • allogenic transplantation the HSCs are obtained from a compatible healthy donor.
  • HSCs can be extracted from bone marrow, peripheral blood, amniotic fluid or umbilical cord blood, wherein extraction from peripheral blood is clinically the most relevant method.
  • HSCs are collected from the blood through a process called apheresis. Prior to apheresis, HSCs need to be recruited from the donor's bone marrow into the peripheral circulation through treatment with Granulocyte-colony stimulation factor (G-CSF). This treatment is expensive and has potential side effects.
  • G-CSF Granulocyte-colony stimulation factor
  • New neurons are generated throughout life in the mammalian hippocampus (Spalding et al., 2013; van Praag et al., 2002). This process, called adult neurogenesis, is critically involved in a variety of hippocampus-dependent forms of learning and memory. In addition, failing or altered neurogenesis has been associated with a number of neuropsychiatric diseases such as major depression, epilepsy, and cognitive aging, suggesting adult hippocampal neurogenesis is relevant for human health and disease (Christian et al., 2014; Kempermann et al., 2008; Scharfman and Hen, 2007).
  • NSPCs neural stem/progenitor cells
  • Large screens have been performed (e.g., by Roche) to identify ways to enhance neurogenic capacity of NSPCs in the aging brain or in disease states where NSPC activity is reduced.
  • proliferating NSPCs have been used as a starting cell type. Given that the vast majority of NSPCs within the adult brain is quiescent (or even dormant), these screens do not recapitulate the in vivo situation. A safe, reliable method for in vivo activation and expansion of quiescent NSCPs would be highly desirable. Description
  • Malonyl-CoA (CAS No. 524-14-1 ) is the coenzyme A derivative of malonic acid (propanedioic acid).
  • a method of hematopoietic stem cell generation comprising the steps of
  • the malonate salt is a sodium or disodium, potassium or dipotassium or mixed sodium potassium salt of malonic acid.
  • the compound is present in a concentration from 50 to 500 ⁇ . In certain embodiments, the compound is present in a concentration from 100 to 200 ⁇ . In certain embodiments, the compound is present in a concentration from 150 to 350 ⁇ . In certain embodiments, the compound is present in a concentration from 100 to 400 ⁇ . In certain embodiments, the compound is present in a concentration of approximately (+- 20 ⁇ ) of 100 ⁇ , 150 ⁇ , 200 ⁇ , 250 ⁇ or 300 ⁇ .
  • the expansion is performed for 3 to 10 days. In certain embodiments, the expansion is performed to effect an approximate 5-fold expansion of the starting cell number.
  • the hematopoietic stem cell isolate is a peripheral blood sample.
  • the hematopoietic stem cell isolate comprises 500 to 4000 CD34+ stem cells per ml, particularly 500 to 2000 CD34+ stem cells per ml, more particularly approx. 1000 CD34+ stem cells per ml.
  • This cell concentration is usually achieved if the donor has not been treated with factors that lead to the mobilization of stem cells from the bone marrow into the peripheral blood (HSC mobilizing factors).
  • HSC mobilizing factors factors that lead to the mobilization of stem cells from the bone marrow into the peripheral blood
  • the obtained stem cells can be expanded according to the first aspect of the invention to levels similar to those obtained from donors that have been treated with HSC mobilizing factors.
  • the method according to the first aspect of the invention thus allows abstaining from treatment of donors with HSC mobilizing factors, while still obtaining sufficient stem cell numbers for transplantation. It is expected that this will increase the willingness of healthy individuals to agree with a peripheral blood stem cell donation.
  • the hematopoietic stem cell isolate comprises 4.000 to 40.000 CD34+ stem cells per ml, particularly 5.000 to 20.000 CD34+ stem cells per ml, more particularly approx. 10.000 to 20.000 CD34+ stem cells per ml.
  • This cell concentration is usually achieved if the donor has been treated with HSC mobilizing factors.
  • the number of cells obtained from donors, even from donors treated with HSC mobilizing factors is not sufficient for stem cell transplantation, which requires approx. 2 x 10 6 CD34+ stem cells per kg body weight. In these cases, several apheresis sessions are required.
  • the method according to the first aspect of the invention thus allows to further expand the stem cell isolated from a donor, making several apheresis sessions unnecessary, while still obtaining sufficient stem cell numbers for transplantation. It is expected that this will increase the willingness of healthy individuals to agree with a peripheral blood stem cell donation.
  • the hematopoietic stem cell isolate is a peripheral blood sample comprising less than 10.000 CD34+ cells per ml, particularly less than 5.000 CD34+ cells per ml, more particularly less than 2.500 CD34+ cells per ml.
  • the hematopoietic stem cell isolate is a peripheral blood sample comprising less than 50.000 CD34+ cells per ml, particularly less than 20.000 CD34+ cells per ml, more particularly less than 10.000 CD34+ cells per ml.
  • malonyl-CoA, malonic acid or a malonate salt is provided for use in treatment / therapy of a condition requiring hematopoietic stem cell transplantation.
  • the stem cell transplantation may be autologous or allogenic.
  • the condition is selected from the group comprising hematological malignancies including leukemias, lymphomas and myelomas; solid tumors including neuroblastoma, Ewing sarcoma and choriocarcinoma; hematologic diseases including myelodysplastic syndromes, anemias and myeloproliferative disorders; amyloidoses; radiation poisoning; viral diseases including HTLV and HIV; lysosomal storage disorders; immunodeficiencies including ataxia teleangiectasia, DiGeorge syndrome, severe combined immunodeficiency and Wiskott-Aldrich syndrome.
  • Treatment of the abovementioned conditions includes chemotherapy or radiation therapy, leading to damage or ablation of the bone marrow.
  • the patients rely on the transplantation of a sufficient amount of potent hematopoietic stem cells to rebuild their hematopoietic system.
  • the method / use of malonyl-CoA or malonic acid or a malonate salt described herein is used in the context of a treatment or therapy of the above mentioned conditions, however the use is not directed towards a therapy of the underlying disease, but to the generation or expansion of the stem cells required to compensate the loss of cells during treatment.
  • malonyl-CoA, malonic acid or a malonate salt is employed for in vitro expansion of hematopoietic stem cells obtained from a donor prior to transplantation.
  • malonyl-CoA, malonic acid or a malonate salt is provided for use in therapy or prevention of neuropsychiatric disorders including major depression, epilepsy, and cognitive aging.
  • a dosage form comprising malonyl- CoA, malonic acid or a malonate salt for use in therapy or prevention of neuropsychiatric disorders.
  • malonyl-CoA malonic acid or a malonate salt for stem cell expansion
  • said stem cell is a hematopoietic stem cell and said expansion occurs in vitro.
  • the inventors present evidence that the regulation of lipid metabolism by malonyl-CoA affects HSC activity. Surprisingly, the inventors show that increasing levels of malonyl-CoA is instructive and sufficient to cause the expansion of murine and human HSCs.
  • said stem cell is a neural stem/progenitor cell (NSPC) and said expansion occurs in vivo.
  • NSPC neural stem/progenitor cell
  • the inventors have used several complementary approaches to show that quiescent NSPCs in the embryonic and adult brain are in a distinct metabolic state that depends on high levels of fatty acid oxidation to maintain quiescence. This state is controlled by levels of malonyl-CoA, which in turn are regulated by the small regulatory protein Spot14. Surprisingly, the inventors show that manipulating levels of malonyl-CoA is instructive and sufficient to change quiescence behavior of NSPCs.
  • a method of treating a condition requiring hematopoietic stem cell transplantation comprising the steps of a. obtaining hematopoietic stem cells from a donor;
  • a preparation of hematopoietic stem cells obtained by a method according to the first aspect of the invention is provided.
  • the preparation of hematopoietic stem cells according to this aspect of the invention is different from an untreated isolated preparation of hematopoietic stem cells with regard to distinct biochemical markers.
  • the preparation of hematopoietic stem cells according to this aspect of the invention is different from preparation of hematopoietic stem cells by previously described methods with regard to stem cell potency, wherein the inventive stem cell preparation exhibits an increased potency.
  • the potency can be determined by (serial) transplantation assays in which the ability of the cells to reconstitute the previously ablated bone marrow stem cell population of a mouse is determined (reconstitution assay).
  • the preparation of hematopoietic stem cells according to this aspect of the invention is different from preparation of hematopoietic stem cells by previously described methods with regard to genetic modifications of the cells.
  • the cells of the inventive hematopoietic stem cell preparation do not comprise any genetic manipulation, in particular no genetic manipulation that results increased proliferative activity of the cells, such as c-myc overexpression.
  • Fig. 1 shows that the cellular composition of quiescent NSPCs is set to allow optimal FAO.
  • B The increase in Cpt1 a mRNA levels is also reflected on protein levels, as revealed by Western blot analysis.
  • C Co-stainings against Cpt1 a and a mitochondrial marker (Mitotracker) reveals the mitochondrial localization of Cpt1 a in quiescent NSPCs. Shown is a representative confocal image.
  • D mRNA levels of the previously described novel quiescence marker Spot14 are highly upregulated in quiescent (quie) NSPCs compared to proliferating (prol) NSPCs.
  • Fig. 2 shows that quiescent NSPCs use FAO for energy purposes and as a carbon source.
  • TCA tricarboxylic acid cycle
  • Fig. 3 shows that manipulating Malonyl-CoA levels is sufficient to prevent NSPC quiescence and to trigger cell cycle re-entry under quiescence condition.
  • A Schematic outline of the experimental setup.
  • B Addition of malonyl-CoA (100 ⁇ or 200 ⁇ ) at the beginning of quiescence induction is sufficient to prevent quiescence entry in a dose dependent manner, as revealed with the cell cycle marker Ki67 and the mitotic marker phospho Histone 3 (pH3). Shown are representative images of indicated doses and the quantification of cycling and proliferating cells after 3 days of quiescence induction (mean ⁇ SEM).
  • Fig. 4 shows that Malonyl-CoA increases numbers of long-term (LT)-and short-term(ST)-HSCs in vitro.
  • A Treatment of LT-HSCs with different doses of Malonyl-CoA (5 ⁇ or 50 ⁇ ) leads to an increase in cell number compared to control treated LT-HSCs (LiCI) after 7d of in vitro expansion. After culturing LT-HSCs in the presence of Malonyl-CoA, there is a significant increase in the number of small cells (5-15 ⁇ diameter), corresponding to the initial cell size after isolation and suggestive of symmetric expansion.
  • Fig. 5 shows that exogenously applied malonyl-CoA is incorporated into new lipids and that increased proliferation upon malonyl-CoA exposure in quiescent NSPCs is at least partially regulated by an increase in FASN-dependent de novo lipogenesis.
  • A Scheme of the experimental procedure to detect whether exogenously applied malonyl-CoA can be used by NSPCs. Radioactively labeled malonyl-CoA (14C-malonyl-CoA) was applied together with non-labelled malonyl-CoA (100 ⁇ ) to proliferating NSPCs for 48h. Intracellular lipids were isolated, separated by thin layer chromatography and their radioactivity was measured by scintillation counts.
  • Fig. 6 shows a surface marker analysis of LKS cells expanded in the presence of malonyl- CoA.
  • A depicts the experimental procedure
  • B shows the flow cytometry analysis of control and malonyl-CoA treated cells.
  • the surface markers LKS (lin-, c-kit+, sca1 +) (left graph), CD150+/CD48- (middle graph) and CD34+/CD34- (right graph) were measured in both treatment groups.
  • C depicts the absolute numbers of LKS, CD150+ and CD34- cells.
  • Fig. 7 shows the experimental setup for the primary LKS transplant.
  • Fig. 8 shows the outcome of the blood cell measurement in the time-course experiment of the primary LKS transplant.
  • Chimerism is a measure of the origin of the blood cells. The higher the percentage indicated the more blood cells of this type originated from the indicated treatment group.
  • (A) depicts the chimerism of all blood cells
  • (B) depicts the chimerism of the myeloid lineage of blood cells.
  • (C) depicts the chimerism of the lymphoid lineage of blood cells.
  • the chimerism of all blood cells and of the lymphoid lineage in the malonyl-CoA treated group differed significantly from the control group (no treatment).
  • Fig. 9 shows the experimental setup of the secondary LKS transplant.
  • Fig. 10 shows the outcome of the blood cell measurement in the time-course experiment of the secondary LKS transplant.
  • Chimerism is a measure of the origin of the blood cells. The higher the percentage indicated the more blood cells of this type originated from the indicated treatment group.
  • (A) depicts the chimerism of all blood cells
  • (B) depicts the chimerism of the myeloid lineage of blood cells.
  • (C) depicts the chimerism of the lymphoid lineage of blood cells.
  • the chimerism of the malonyl-CoA treated group tended to be higher than the control group, but the difference was not statistically different.
  • Cptl a a rate-limiting enzyme of FAO we found to be upregulated in quiescent NSPCs using unbiased proteomics.
  • the inventors next aimed to understand the molecular mechanism underlying high levels of FAO in quiescent NSPCs.
  • the inventors have previously shown that Spot14 is selectively expressed in quiescent NSPCs in vivo (Knobloch et al., 2013, Nature 493, 226-230; Knobloch et al., 2014, Stem Cell Reports 3, 735-742).
  • quantitative reverse transcriptase PCR showed more than 30-fold upregulation of Spot14 mRNA upon quiescence induction in vitro, again reversed upon re-exposure to proliferation conditions (Figure 1 D).
  • the inventors next used 13 C-labeled palmitic acid in combination with mass spectrometry to trace the incorporation of the labeled carbon atoms in quiescent vs. proliferative NSPCs (Figure 2B).
  • the inventors found highly significant increases in the incorporation of 13 C into TCA intermediates as well as into proteins derived from TCA intermediates in quiescent NSPCs ( Figure 2C-D). Taken together, these data indicate that quiescent NSPCs use FAO for energy purposes as well as an alternative carbon source for amino acid biosynthesis.
  • LKS cells were isolated from CD45.1 donor mice and cultured in the presence of malonyl-CoA (1 ⁇ ) for 7 days (Fig. 6 A). After 7 days the surface markers of ST-HSC (lin-, c-kit+, Seal +, CD48-, CD150+, CD34+) and LT-HSC (lin-, c-kit+, Sca1 +, CD48-, CD150+, CD34-) were analysed by flow cytometry (Fig. 6 B). The number of LKS cells, CD150+ and CD34- cells was significantly increased in the group of malonyl-CoA treated cells as compared to untreated controls (Fig. 6 C).
  • Potency of the cells expanded according to the method of the present invention was tested in vivo, to assess if the method is associated with a loss in potency.
  • LKS cells were isolated from CD45.1 donor mice and cultured in the presence of malonyl-CoA (100 ⁇ ) for 7 days (Fig. 7).
  • Cultured LKS cells (20.000 cells/mice) were combined with 350.000 total bone marrow cells from CD45.1/2 competitor mice and injected into irradiated CD45.2 recipient mice (850Gy, split dose).
  • Blood samples were analysed for myeloid and lymphoid blood cells as well as total blood cell count in a time course experiment after 4, 8, 12, 16, 20 and 24 weeks (Fig. 8).
  • Total blood cells and lymphoid blood cells were significantly elevated in the group of malonyl-CoA treated LKS cells (Fig. 8 A, C) whereas myeloid cells showed only a slight increase in the malonyl-CoA treated group (Fig. 8 B). This demonstrates the usability of the malonyl-CoA treated cells in a primary transplant in vivo.
  • a secondary LKS transplant In a second step, the longterm effect of malonyl-CoA treatment was evaluated in a secondary LKS transplant. Towards these ends total bone marrow cells of the CD45.2 recipient mice from the primary LKS transplant described above were isolated after the end of the time course experiment and injected into irradiated CD45.2 mice (850Gy, split dose) (Fig. 9). Blood samples were analysed for myeloid and lymphoid blood cells as well as total blood cell count in a time course experiment after 4, 8, 12, 16, 20 and 24 weeks (Fig. 10).
  • mice were kept with littermates under a 12h dark/light cycle in single ventilated cages and with ad libitum access to food and water.
  • the Cpt1a-EGFP reporter mouse line (STOCK Tg(Cpt1 a- EGFP)IP41 Gsat/Mmucd) was generated by the Mutant Mouse Regional Resource Centers (MMRRC). Time-mated C57/BI6 female mice were obtained from Janvier Labs (France). All animal experiments were performed according to Swiss regulatory standards and approved by the Veterinary office of the Canton of Zurich.
  • Cptl a shRNA sequences were designed using the RNAi Consortium hairpin candidate sequences selection (www.broadinstitute.org/rnai/trc) against mouse Cptl a.
  • the shRNA knockdown constructs (derived from Lentil_ox3.7) were cloned to express mCherry under the CMV promoter and shRNAs under the U6 promoter. Knockdown efficiency was tested in transfected mouse liver hepatoma cells.
  • NSPCs were plated as described above. 4-6 adjacent areas per well were imaged every 4h on a heated and C02-controlled inverted microscope over the indicated time. Stitched phase contrast images were analysed using ImageJ. Several processing steps (bandpass filtering, Gaussian blurs, thresholding) were used to automatically analyze the area covered by cells. Proteomic analysis
  • Proteins were extracted from proliferating and quiescent NSPCs and samples were processed using a modified protocol by filter aided sample preparation (FASP) (Wisniewski et al., 2009, Nature methods 6, 359-362), followed by Solid Phase Extraction (SPE) C18 clean up. All data was acquired on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific), which was connected to an Easy-nLC 1000 HPLC system (Thermo Scientific). Raw files were processed with Progenesis Ql for proteomics (Nonlinear Dynamics). Mascot (2.4.1 ) was used for searching a target-decoy mouse database downloaded from uniprot (03/01/2015).
  • FASP filter aided sample preparation
  • SPE Solid Phase Extraction
  • Radioactively labeled 3 H-palmitic acid and 14 C-palmitic acid were purchased from Perkin Elmer. FAO was assessed by the production and release of tritiated water or 14 C02 as previously described (Djouadi et al., 2003, Mol Genet Metab 78, 1 12-1 18; Huynh et al., 2014, Methods in enzymology 542, 391 -405). In brief, proliferating and quiescent NSCPs were incubated with labeled palmitic acid for 2.5h or 4h and medium was collected for subsequent processing. The amount of radioactive 3 H20 or 14 C02 generated was measured by scintillation counting. All measurements were normalized to protein content.
  • mice were transcardially perfused with 0.9% saline solution followed by 4% PFA solution. Brains were processed as previously described for immunohistology (Knobloch et al., 2013, Nature 493, 226-230). Staining was performed on 40 ⁇ thick free-floating sections. Primary antibodies were incubated overnight at 4 °C, followed by secondary antibody incubation for several hours at room temperature (RT). Cells were fixed with 4% PFA and antibody stainings were done overnight at 4 °C, secondary antibodies were incubated 2-4 h at RT. Antibody details are available in the supplemental information. Image acquisition and analysis
  • RNA of proliferating, quiescent, replated proliferating and formerly quiescent NSPCs was isolated and processed as described before (Knobloch et al., 2013, Nature 493, 226-230).
  • Taqman probes against Cptl a, Spot14 and ⁇ -actin and RT-PCR master-mix were used according to the manufacturer's protocol.
  • Real time PCR and data analyses were performed on an Applied Biosystems 7900HT System. Fold changes were calculated using the deltadelta Ct methods.
  • Protein lysates of proliferating and quiescent NSPCs were separated by SDS-PAGE electrophoresis followed by transfer to PVDF membrane. Membranes were incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies for several hours at RT. The signal was revealed by enhanced chemiluminescence substrate and quantification was done with ImageJ.
  • Metabolites of proliferating and quiescent NSPCs were extracted with cold acetonitrile:methanol:water solvent (40:40:20). Extracts were dried, re-suspended in water and analyzed by LC-MS/MS on a Thermo Quantum Ultra instrument equipped with a Waters Acquity UPLC (Buescher et al., 2010, Anal Chem 82, 4403-4412). Data analysis was performed using own software written in Matlab (The Mathworks). Measured malonyl-and acetyl-CoA values were normalized to the average cell number.
  • Proliferating and quiescent NSPCs were incubated for 24h with the corresponding medium containing 100 ⁇ 13 C-labelled palmitate (Cambridge Isotope Laboratories Inc.). Metabolites were extracted with 80% methanol. Samples were processed as described in the supplemental information and GC-MS analyses were performed using an Agilent 7890A GC equipped with a HP-5 ms 5% Phenyl Methyl Silox capillary column, interfaced with a triple quadruple tandem mass spectrometer (Agilent 7000B, Agilent Technologies). The GC-MS analyses were performed in Single Ion Monitoring (SIM) scanning for the isotopic pattern of metabolites.
  • SIM Single Ion Monitoring
  • HSC experiments Cells were isolated from hindlegs and hips of 12-week old BI6 mice according to a previously described protocol (Roch et al, Stem Cells 2015). In brief, bones were crushed in PBS-EDTA, red blood cells were lysed and lineage depletion was performed using a biotin-mouse lineage cocktail (BD Biosciences) combined with magnetic bead sorting (autoMACS, BD Biosciences). Cells were stained with Strep-PO (Life Technologies), cKit-PECY7 (BioLegend), Sca1 -APC (BioLegend), CD150 PECY5 (BioLegend), CD48-PB (BioLegend), CD34-FITC (eBioscience) and PI (Fluka).
  • Strep-PO Life Technologies
  • cKit-PECY7 BioLegend
  • Sca1 -APC BioLegend
  • CD150 PECY5 BioLegend
  • CD48-PB BioLegend
  • the LT-HSC population was defined as lineage depleted (lin neg), C-Kit+, Sca1 +, CD48-, CD150+, CD34-.
  • the ST-HSC population was defined as lineage depleted (lin neg), C-Kit+, Sca1 +, CD48-, CD150+, CD34+.
  • 80 cells per well and condition were sorted on a FACS Aria II flow cytometer (BD Biosciences) into round bottom well plates containing 200 ⁇ basal medium supplemented with Stemline II (Sigma-Aldrich), 100 ng/ml stem cell factor (SCF) and 2 ng/ml Flt-3 ligand as previously described (Roch et al, Stem Cells 2015).

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Abstract

La présente invention concerne un procédé de production de cellules souches hématopoïétiques, comprenant les étapes consistant à utiliser un isolat de cellules souches hématopoïétiques prélevé chez un donneur et à procéder à l'expansion desdites cellules souches hématopoïétiques in vitro en présence d'un composé sélectionné parmi l'acide malonique, la malonyl-coenzyme A et un sel de malonate. L'invention concerne en outre l'utilisation de malonyl-coenzyme A, d'acide malonique ou d'un sel de malonate dans l'expansion de cellules souches et dans le traitement de troubles et de maladies neuropsychiatriques nécessitant une greffe de cellules souches hématopoïétiques.
PCT/EP2018/056766 2017-03-17 2018-03-16 Procédé d'expansion in vitro de cellules souches WO2018167317A1 (fr)

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WO2004011621A2 (fr) * 2002-07-29 2004-02-05 Es Cell International Pte Ltd. Procede en plusieurs etapes de differenciation des cellules sensibles a l'insuline positive, au glucose
WO2008073748A1 (fr) * 2006-12-08 2008-06-19 University Of Rochester Expansion de cellules souches hématopoïétiques
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