scholarly journals Synergistic Effects of Combined Nurr1 Overexpression and Natural Inducers on the More Efficient Production of Dopaminergic Neuron-Like Cells From Stem Cells

2022 ◽  
Vol 15 ◽  
Author(s):  
Reyhaneh Beiki ◽  
Mahsa Khaghani ◽  
Fariba Esmaeili ◽  
Fariba Dehghanian
Keyword(s):  
Stem Cells ◽  
Dopaminergic Neuron ◽  
Synergistic Effects ◽  
Neonatal Rat ◽  
Cresyl Violet ◽  
Brain Extract ◽  
Gene Overexpression ◽  
Efficient Production ◽  
Differentiation Potential ◽  
Differentiated Cells

The development of dopaminergic (DA) neurons is a very complex process, and a combination of extrinsic and intrinsic factors involves their differentiation. Transcription factor, Nurr1 plays an essential role in the differentiation and maintenance of midbrain DA neurons. Nurr1-based therapies may restore DA function in Parkinson's disease (PD) by replacing damaged cells with differentiated cells derived from stem cells. Providing tissue-specific microenvironments such as brain extract can effectively induce dopaminergic gene expression in stem cells. The present study aimed to investigate the combined effects of Nurr1 gene overexpression and a neonatal rat brain extract (NRBE) induction on dopaminergic differentiation of P19 stem cells. In order to neural differentiation induction, stably Nurr1-transfected cells were treated with 100 μg/ml of NRBE. The differentiation potential of the cells was then evaluated during a period of 1–3 weeks via various methods. The initial evaluation of the cells by direct observation under a light microscope and cresyl violet specific staining, confirmed neuron-like morphology in the differentiated cells. In addition, different molecular and cellular techniques, including real-time PCR, immunofluorescence, and flow cytometry, demonstrated that the treated cells expressed pan-neuronal and dopaminergic markers. In all experimental groups, neuronal phenotype with dopaminergic neuron-like cells characteristics mainly appeared in the second week of the differentiation protocol. Overall, the results of the present study revealed for the first time the synergistic effects of Nurr1 gene overexpression and possible soluble factors that existed in NRBE on the differentiation of P19 stem cells into dopaminergic neuron-like cells.

scholarly journals PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

2016 ◽  
Vol 113 (16) ◽  
pp. E2306-E2315 ◽  
Author(s):  
Vashe Chandrakanthan ◽  
Avani Yeola ◽  
Jair C. Kwan ◽  
Rema A. Oliver ◽  
Qiao Qiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Growth Factor ◽  
Tissue Regeneration ◽  
Somatic Cells ◽  
Dependent Manner ◽  
Cell Plasticity ◽  
Differentiation Potential ◽  
Multipotent Stem Cells ◽  
Differentiated Cells

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

scholarly journals Regulation of CXCR6 Expression on Adipocytes and Osteoblasts Differentiated from Human Adipose Tissue-Derived Mesenchymal Stem Cells

2020 ◽  
Vol 2020 ◽  
pp. 1-14
Author(s):  
Seung-Cheol Lee ◽  
Yoo-Jung Lee ◽  
Min Kyoung Shin ◽  
Jung-Suk Sung
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Cell Activation ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Human Mesenchymal Stem Cells ◽  
De Novo Synthesis ◽  
Differentiation Potential ◽  
Differentiated Cells

Human mesenchymal stem cells derived from adipose tissue (hADMSCs) are a desirable candidate in regenerative medicine. hADMSCs secrete growth factors, cytokines, and chemokines and also express various receptors that are important in cell activation, differentiation, and migration to injured tissue. We showed that the expression level of chemokine receptor CXCR6 was significantly increased by ~2.5-fold in adipogenic-differentiated cells (Ad), but not in osteogenic-differentiated cells (Os) when compared with hADMSCs. However, regulation of CXCR6 expression on hADMSCs by using lentiviral particles did not affect the differentiation potential of hADMSCs. Increased expression of CXCR6 on Ad was mediated by both receptor recycling, which was in turn regulated by secretion of CXCL16, and de novo synthesis. The level of soluble CXCL16 was highly increased in both Ad and Os in particular, which inversely correlates with the expression on a transmembrane-bound form of CXCL16 that is cleaved by disintegrin and metalloproteinase. We concluded that the expression of CXCR6 is regulated by receptor degradation or recycling when it is internalized by interaction with CXCL16 and by de novo synthesis of CXCR6. Overall, our study may provide an insight into the molecular mechanisms of the CXCR6 reciprocally expressed on differentiated cells from hADMSCs.

Purinergic Stimulation of Human Bone Marrow-Derived Mesenchymal Stem Cells Modulate Their Function and Differentiation Potential.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3848-3848
Author(s):  
Marilena Ciciarello ◽  
Valentina Salvestrini ◽  
Davide Ferrari ◽  
Sara Gulinelli ◽  
Roberta Zini ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Transcription Factor ◽  
Human Bone ◽  
Differentiation Potential ◽  
Qrt Pcr ◽  
Differentiated Cells

Abstract Abstract 3848 Introduction: Human bone marrow derived Mesenchymal Stem Cells (hMSCs) are adult multipotent cells. hMSCs differentiate in vitro and in vivo into several tissue lineages originating from the three germinal layers making them attractive candidates for bioengineering and cellular therapy. Thus, it seems of great relevance to search putative messengers and signalling able to modulate their proliferation and differentiation. Nucleotides triphosphates are extracellular messengers binding to specific receptors (P2Rs) that modulate cell functions depending on the cell type. Controversial information is available on P2 expression and activity in hMSCs. Methods and Results: Here we found that hMSCs expressed several P2R subtypes. hMSCs were very resistant to the cytotoxic effects of high concentrations of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP treatment down-regulated cell proliferation and up-regulated cell migration genes in hMSCs. Functional studies confirmed the inhibitory activity of ATP on proliferation and clonogenic ability of hMSCs. Furthermore, ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, xenotransplant experiments showed that the homing capacity of hMSCs to murine bone marrow was increased by ATP pre-treatment. Moreover, ATP increased pro-inflammatory cytokines production (IL-2, IFN-g, IL-12p70), while decreased secretion of the anti-inflammatory cytokine IL-10. This finding was associated with the reduced ability of ATP-treated hMSC of inhibiting T-cell proliferation. Microarrays data suggested that several genes implicated in hMSC differentiation can be modulated by ATP treatment. To further investigate this issue, hMSCs cells were cultured under adipogenic or osteogenic conditions and were transiently exposed to ATP before starting differentiation or continuously exposed to ATP for the first 3 days of differentiation induction. We demonstrated that adipogenesis-related accumulation of lipids, analyzed by Oil red O staining, was more evident in ATP treated cultures. Furthermore, quantitative real time PCR (qRT-PCR) assay showed that mRNA expression of PPARg, a transcription factor early up-regulated during adipogenesis, was significantly increased in hMSCs differentiated cells treated with ATP. In osteogenic condition, analysis of mineralized area through Alizarin Red staining, indicated that ATP treatment enhanced the extent of mineralization compared to untreated control. The expression of RUNX2, a key transcription factor in osteogenesis, analyzed by qRT-PCR in differentiated cells confirmed data obtained in Alizarin-based assay. Conclusions: These data demonstrated that purinergic signalling modulates biological functions and differentiation potential of hMSCs. Disclosures: No relevant conflicts of interest to declare.

Abstract 155: C-kit+ Cardiac Progenitor Cells Contain a Subpopulation of Slowly Adherent Cells With Higher Cardiomyogenic and Pro-angiogenic Profile

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Shahab Ghafghazi ◽  
Rakesh Ponnapureddy ◽  
Mitesh Solanki ◽  
Sorabh Sharma ◽  
Pramod Kayathi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Umbilical Vein ◽  
Cardiac Regeneration ◽  
Neonatal Rat ◽  
Cell Surface Receptor ◽  
Adherent Cells ◽  
Differentiation Potential ◽  
Rat Cardiomyocytes ◽  
Angiogenic Potential ◽  
Isolation Methods

Background: Isolation of a pure population of cardiomyogenic cardiac stem cells (CPCs) has been the Holy-Grail in cardiac regeneration. C-kit, a cell surface receptor, has been proposed as a marker of CPCs and the result of multiple preclinical studies and a phase I clinical trial has been encouraging. Nonetheless, optimization of isolation methods is desirable given the heterogeneity of c-kit positive CPCs. We hypothesize that isolation of c-kit positive CPCs based on differential adhesion identifies a superior population of cells. Methods: Non-adherent cells from digested murine hearts were transferred to successive flasks at 2, 4, 24, 48 and 72 hours. Hence, 5 fractions of cells were isolated based on their adhesion profile and labeled rapidly-adherent (1 and 2) and slowly-adherent (3 to 5). Cells were then expanded and sorted based on c-kit expression and expanded to seven passages. Cells were analyzed for surface markers and cardiomyogenic and pluripotent expression profile by flow-cytometry and real-time polymerase chain reaction. Differentiation potential of the two fractions was examined and their paracrine profile compared using human umbilical vein endothelial cells and neonatal rat cardiomyocytes. Results: C-kit expression was maintained in the slowly-adherent fraction while precipitously dropping in the rapidly-adherent one. In addition, the former expressed lower levels of mesenchymal/fibroblast (CD90.2) and macrophage (CD11b/CD45) markers and higher levels of pluripotent (Oct-4, Nanog, Dppa-3, Rif) and cardiac markers (Nkx2.5, Gata4, Mybpc3 and cTnI) comparing to the latter. Finally, slowly-adherent CPCs had superior paracrine profile compared to rapidly-adherent one. Conclusion: C-kit positive CPCs cells isolated from murine myocardium are heterogeneous and better isolation methods are required to enrich a purer population of cardiomyogenic stem cells. In that vein, isolation of c-kit positive CPCs based on their adhesion profile identifies a subpopulation of cells capable of maintaining c-kit positivity and superior in terms of cariomyogenic and pro-angiogenic potential.

52 EFFECT OF DONOR CELLS WITH VARIOUS DIFFERENTIATED STATUS ON THE DEVELOPMENTAL COMPETENCE OF PORCINE NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 144
Author(s):  
J. G. Kim ◽  
E. J. Kang ◽  
M. K. Kim ◽  
S. Y. Choe ◽  
G. J. Rho
Keyword(s):  
Stem Cells ◽  
Nuclear Transfer ◽  
Statistical Significance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Differentiation Potential ◽  
Developmental Potential ◽  
Differentiated Cells ◽  
Donor Cells ◽  
Fetal Fibroblasts

Adult stem cells are more desirable than somatic cells for nuclear transfer (NT) because of their easy reprogrammability to resemble the genome of the zygote (Zhu et al. 2004 Biol. Reprod. 70, 1088–1095). Mesenchymal stem cells (MSCs) are a heterogeneous population of uncommitted and lineage-committed cells and have a more flexible potential as donor cells for NT. The aim of this study was to compare the developmental potential of NT embryos using undifferentiated (MSCs) and differentiated cells in the same lineage (osteocyte, adipocyte, and chondrocyte) by assessing the cleavage and blastocyst rates. Fetal fibroblasts were used as NT control. MSCs obtained from the aspirated bone marrow of a neonatal pig were cultured in advanced-DMEM (ADMEM) supplemented with 5% FCS. The differentiation potential was demonstrated by culture of MSCs at passage 3 under the conditions that were favorable for adipogenic, osteogenic, and chondrogenic development (Pittenger et al. 1999 Science 284, 143–147). For NT, cells from passages 3–5 were transferred into the perivitelline space of enucleated MII oocytes that had been in vitro-matured after collection from slaughterhouse-derived ovaries. After fusion with a needle-type electrode, eggs were cultured in 7.5 µg mL−1 cytochalasin B for 3 h, and subsequently cultured in PZM-3 medium for 6 days. Statistical significance was tested using ANOVA with Bonferroni and Duncan tests. The results are presented in Table 1. The rates of cleavage and development to blastocyst stage of NT embryos varied among donor cell sources. Most eggs (92.2 ± 2.7%) cloned with MSCs cleaved, and 47.8% of eggs developed to the blastocyst stage. In contrast, NT eggs using differentiated MSCs—osteocytes, adipocytes, chondrocytes, and controls (fetal fibroblasts)—revealed significantly (P < 0.05) lower cleavage (74.5, 63.4, 74.3, and 66.4%, respectively) and blastocyst development (33.7, 30.1, 36.5, and 25.5%, respectively) rates than those using undifferentiated MSCs. The results demonstrate that the genome of donor cells with different differentiated status supports embryonic development to various degrees, and multipotent MSCs might have a greater potential in producing viable cloned porcine embryos. Table 1.Development of NT embryos with undifferentiated and differentiated cells This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

scholarly journals BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells

2020 ◽  
Vol 3 (5) ◽  
pp. e201900534 ◽  
Author(s):  
Cristina Ameneiro ◽  
Tiago Moreira ◽  
Alejandro Fuentes-Iglesias ◽  
Alba Coego ◽  
Vera Garcia-Outeiral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Energy Metabolism ◽  
Cell Fate ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Loss Of Function ◽  
Differentiation Potential ◽  
Differentiated Cells ◽  
Permanent Loss

BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.

Putative germline and pluripotent stem cells in adult mouse ovary and their in vitro differentiation potential into oocyte-like and somatic cells

Zygote ◽  
2017 ◽  
Vol 25 (3) ◽  
pp. 358-375 ◽  
Author(s):  
Yashar Esmaeilian ◽  
Arzu Atalay ◽  
Esra Erdemli
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Adult Mouse ◽  
Immunofluorescence Staining ◽  
Neural Cells ◽  
Stem Cell Markers ◽  
Rt Pcr ◽  
Mouse Ovary ◽  
Differentiation Potential ◽  
Differentiated Cells

SummaryAccording to classical knowledge of reproductive biology, in the ovary of female mammals there is a limited number of oocytes and there is no possibility of renewal if the oocytes are lost due to disease or injury. However, in recent years, the results of some studies on renewal and formation of oocytes and follicles in the adult mammalian ovary have led to the questioning of this opinion. The aim of our study is to demonstrate the presence of putative germline and pluripotent stem cells in the adult mouse ovary and their differentiation potential into germ and somatic cells. In ovary tissues and cells harvested from pre-differentiation step, the expression of pluripotent and germline stem cell markers was analysed by reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence staining and western blotting. Embryoid bodies that formed in this step were analysed using immunofluorescence staining and transmission electron microscopy. Ovarian stem cells were induced to differentiate into oocyte, osteoblast, chondrocyte and neural cells. Besides morphological observation, differentiated cells were analysed by RT-PCR, histochemical and immunofluorescence staining. Expression of germline and pluripotent stem cell markers both in mRNA and at the protein level were detected in the pre-differentiated cells and ovary tissues. As a result of the differentiation process, the formation of oocyte-like cells, osteoblasts, chondrocytes and neural cells was observed and characteristics of differentiated cells were confirmed using the methods mentioned above. Our study results revealed that the adult mouse ovary contains germline and pluripotent stem cells with the capacity to differentiate into oocyte-like cells, osteoblasts, chondrocytes and neural cells.

scholarly journals Stem Cells as New Agents for the Treatment of Infertility: Current and Future Perspectives and Challenges

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Vladislav Volarevic ◽  
Sanja Bojic ◽  
Jasmin Nurkovic ◽  
Ana Volarevic ◽  
Biljana Ljujic ◽  
...  
Keyword(s):  
Stem Cells ◽  
Reproductive Medicine ◽  
Current Knowledge ◽  
Building Blocks ◽  
Future Perspectives ◽  
Differentiation Potential ◽  
New Agents ◽  
Differentiated Cells ◽  
Undifferentiated Cells

Stem cells are undifferentiated cells that are present in the embryonic, fetal, and adult stages of life and give rise to differentiated cells that make up the building blocks of tissue and organs. Due to their unlimited source and high differentiation potential, stem cells are considered as potentially new therapeutic agents for the treatment of infertility. Stem cells could be stimulatedin vitroto develop various numbers of specialized cells including male and female gametes suggesting their potential use in reproductive medicine. During past few years a considerable progress in the derivation of male germ cells from pluripotent stem cells has been made. In addition, stem cell-based strategies for ovarian regeneration and oocyte production have been proposed as future clinical therapies for treating infertility in women. In this review, we summarized current knowledge and present future perspectives and challenges regarding the use of stem cells in reproductive medicine.

scholarly journals Stem cell properties, current legal status and medical application

2017 ◽  
Vol 71 (0) ◽  
pp. 0-0 ◽  
Author(s):  
Ilona Szabłowska-Gadomska ◽  
Leonora Bużańska ◽  
Maciej Małecki
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Legal Status ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Medical Application ◽  
Differentiation Potential ◽  
Differentiated Cells ◽  
Induced Pluripotent

Stem cells due to their unique properties of self-renewal and differentiation play a potential role in the process of damaged tissue repair. Isolated from the inner cell mass of the blastocyst have pluripotential properties and are called embryonic stem cells (ESC). Pluripotential stem cells can be also generated from the differentiated cells by the process of reprogramming and are called induced pluripotent stem cells (iPSC). Stem cells isolated from tissues (somatic or adult stem cells) are more restricted in their differentiation potential and referred as multipotent. The rapid rise in number of clinical trials using somatic stem cells is due to their proved in basic and preclinical studies therapeutic safety and paracrine properties to modulate microenvironment. Increased translation to the clinic of studies using adult stem cells provide hope for patients with diseases for which traditional medicine is powerless .or ineffective. On the other hand progress in iPSC technology allows to derive disease models and personalize future clinical diagnosis and treatment. This paper will focus on characteristics of stem cells, potential application in regenerative medicine, and the current legal status of cell therapy.

A Possible Role for Wnt3a in Enhancing StemRegenin1-Mediated Expansion of Hematopoietic Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4130-4130
Author(s):  
Lucia Duinhouwer ◽  
Elwin Rombouts ◽  
Nesrin Tüysüz ◽  
Jan Spanholtz ◽  
Derk ten Berge ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cell Biology ◽  
Ex Vivo ◽  
Synergistic Effects ◽  
Fold Increase ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 4130 Insufficient engraftment after umbilical cord blood stem cell transplantation (UCB-SCT) may be improved by the administration of ex vivo expanded UCB-derived hematopoietic stem cells (HSC). Although culturing HSC with cytokines such as SCF, Flt3L and TPO results in robust proliferation, it is accompanied with extensive differentiation and loss of self renewal capacity. Inhibition of the Aryl hydrocarbon Receptor (AhR) leads to potent expansion of hematopoietic progenitor cells (HPC) (Boitano et al. Science 2010). Reportedly, Wnt3a inhibits differentiation in different types of stem cells including hematopoietic stem cells(Reya et al. Nature 2003, Ten Berge et al. Nature Cell Biology 2011). Here, we evaluated possible additive or synergistic effects of combining the AhR antagonist StemRegenin1 (SR1) with recombinant purified Wnt3a to expand hematopoietic stem cells ex vivo. UCB-derived CD34-selected cells were cultured in serum-free Glycostem Basal Growth Medium (GBGM) supplemented with the early-acting growth factors SCF, Flt3L and TPO (SFT medium) with or without the addition of SR1 and Wnt3a. Cell number, viability and subset composition within the CD34+ cells were measured using flowcytometry. The multilineage differentiation potential and self renewal capacity of expanded CD34+HPC were evaluated in stroma-supported long-term culture-initiating cell (LTC-IC) assays. Culturing CD34+ cells in SFT medium resulted in a mean 10.2-fold increase in CD34+ cells after 1 week of culture (n=3). Addition of SR1 to the SFT-medium resulted in a 16-fold increase of CD34+ input cells within 7 days, while on the other hand, addition of Wnt3a alone to the SFT-medium resulted in a 7-fold increase in CD34+ cells. However, combining SR1 and Wnt3a in the SFT medium resulted in a 20-fold expansion of CD34+ cells compared to input. The early additive effect of Wnt3a on SFT+SR1-induced expansion of CD34+ cells, however, disappeared upon prolonged culture up to 2–3 weeks. Approximately 3–10% of UCB-derived CD34+ cells could be characterized as phenotypic HSC, as was defined by CD34+CD38lowCD45RAlowCD90+ cells. After culture, we sorted different CD34+-populations to evaluate their functional capacity. Evaluation of LTC-IC frequencies yielded a frequency of 1/23 LTC-IC in the phenotypic HSC-subset (CD34+CD38lowCD45RAlowCD90+) after 28 days of culture in SFT medium with SR1. However, no LTC-IC appeared to be present in the multipotent progenitor subset (MPP, CD34+CD38lowCD45RAlowCD90low). These results indicate that phenotypic HSC maintain their functional LTC-IC capacity after expansion culture. Collectively, our results confirm that SR1 expands HSC with preservation of self-renewal capacity and ability to differentiate into various hematopoietic lineages. In addition, we show that Wnt3a initially enhances SFT+SR1-driven expansion of CD34+ HPC, but reduces the increase in number of CD34+ cells at later stages of culture. These data may suggest that the period of expansion needed for clinical application may be shortened by combining SR1 and Wnt3a. Disclosures: Spanholtz: Glycostem Therapeutics: Employment. Groenewegen:Glycostem Therapeutics: Equity Ownership.

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