scholarly journals SMALL Molecules Mediated Hematopoietic STEM and Progenitor CELLS Expansion for GENE Editing Application

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5803-5803
Author(s):  
Abisha Crystal C ◽  
Saravanabhavan Thangavel ◽  
Shaji Ramachandran Velayudhan ◽  
Alok Srivastava ◽  
Aneesha Nath ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Migration ◽  
Hematopoietic Stem Cells ◽  
Small Molecules ◽  
Gene Editing ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Hematopoietic Stem ◽  
Colony Forming Cell ◽  
Ex Vivo Culture

Abstract Genome editing of Hematopoietic stem Cells has revolutionized the treatment strategies for genetic disorders. Despite this, it still remains a great challenge as hematopoietic stem cells tend to lose its stem-ness during the ex vivo culture and gene editing process. The need for large dose of CD34+ HSPCs for manipulation makes it a seemingly difficult strategy. Recent works suggest that the potential effects of small molecules in expanding cord blood HSPCs ex vivo promoting self-renewal and delaying differentiation. We screened several reported small molecules to identify a condition that promotes the expansion of adult HSPCs for gene manipulation process. The mobilized Peripheral blood HSPCs are purified and cultured with a cytokine cocktail. Along with the cytokine cocktail, we tested several small molecules and in different combinations. Expression of cell surface receptors were analysed by FACS after 12 days of ex vivo culture. Our screening identified a unique culture condition that expanded the primitive stem cell population (CD34+/CD133+/CD90+cells) along with the early progenitors (CD34+/CD133+) and the progenitors (CD34+). Our culture conditions expanded the primitive cells by 20 folds compared to the mock treated cells. Our treatment release experiments suggested that the expansion is due to our culture conditions and are reversible.The colony forming cell (CFC) assay showed about 30 fold increase in the numbers of multilineage colony forming cell (CFU-GEMM) thereby ensuring the proliferation and differentiation capacity of expanded HSPCs. Their differentiation ability was also confirmed by ex vivo differentiation into Megakaryocytes. Our treatment conditions reduced the apoptosis rate during the ex vivo culture and improved their cell migration response towards SDF. The reduced reactive oxygen species levels and increased CXCR4 expression were observed in our expanded HSPCs and these might be the possible reasons for the low apoptosis and better cell migration respectively. Disclosures No relevant conflicts of interest to declare.

scholarly journals Integrin-αvβ3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 83-94 ◽  
Author(s):  
Terumasa Umemoto ◽  
Masayuki Yamato ◽  
Jun Ishihara ◽  
Yoshiko Shiratsuchi ◽  
Mika Utsumi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Matrix Protein ◽  
Ex Vivo ◽  
Extracellular Matrix Protein ◽  
Αvβ3 Integrin ◽  
Hematopoietic Stem ◽  
Β3 Integrin ◽  
Ex Vivo Culture

AbstractThroughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin “inside-out” signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent “outside-in” signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

scholarly journals Combination of SB431542, Chir9901, and Bpv as a Novel Supplement in the Culture of Umbilical Cord Blood Hematopoietic Stem Cells

2020 ◽  
Author(s):  
Elaheh Afzal ◽  
Morteza Zarrabi ◽  
Mohammad Hassan Asghari ◽  
Marzieh Ebrahimi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Small Molecules ◽  
Small Molecule ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract Background: Small molecule compounds have been well recognized for their promising power in generation, expansion and maintenance of embryonic or adult stem cells. The aim of this study was to identify a novel combination of small molecules in order to optimize the ex vivo expansion of umbilical cord blood derived-CD34+ cells. Methods: Considering the most important signaling pathways involved in the self-renewal of hematopoietic stem cells, CB-CD34+ cells were expanded with cytokines in the presence of seven small molecules including SB, PD, Chir, Bpv, Pur, Pμ and NAM. Eliminativism approach was used to find the best combination of selected small molecules for effective ex vivo expansion of CD34+ cell. In each step, proliferation, self-renewal, and clonogenic potential of the expanded cells as well as expression of some hematopoietic stem cell related genes were studied. Finally, the engraftment potential of expanded cells was also examined by the mouse intra-uterine transplantation model. Results: Our data shows that simultaneous use of SB431542 (TGF-β inhibitor), Chir9901 (GSK3 inhibitor) and Bpv (PTEN inhibitor), resulted in a 50-fold increase in the number of CD34+CD38- cells. This was further reflected in approximately 3 times increase in clonogenic potential of the small molecule cocktail-expanded cells. These cells, also, showed a 1.5-fold higher engraftment potential in the peripheral blood of NMRI model of in utero transplantation. These results are in total conformity with up-regulation of HOXB4, GATA2 and CD34 marker gene as well as CXCR4 homing gene. Conclusion: Taken together, our findings introduce a novel combination of small molecules to improve the yield of existing protocols used in the expansion of hematopoietic stem cells.

Preclinical Development of a Cryopreservable Megakaryocyte Cell Product from Cord Blood Derived Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3859-3859
Author(s):  
Helen Fong ◽  
Goar Mosoyan ◽  
Ami Patel ◽  
Ronald Hoffman ◽  
Jay Tong ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Shelf Life ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Hematopoietic Stem ◽  
Cell Product ◽  
Cd34 Cells

Abstract Platelet (PTL) transfusions are currently the most effective treatment for patients with thrombocytopenia. Demand for PTL transfusions has steadily increased in recent years, straining a PTL supply that is already limited due to dependency on volunteer donors, short shelf life, risk of infections, and alloimmunization. This dilemma has stimulated the search for alternative approaches for generating PTLs ex vivo from different sources of hematopoietic stem cells (HSCs). Although PTLs have been successfully generated in cultures initiated with primary human CD34+ cells and pluripotent stem cells, the generation of a clinically relevant PTL product ex vivo faces significant obstacles due to scalability, reproducibility and shelf life. We propose an alternative approach to overcome such obstacles by developing a cryopreservable cell product consisting of megakaryocytes (MK) that can produce PTL in vivo after transfusion into patients. Umbilical cord blood units (CBU) are FDA-approved, readily available sources for allogeneic HSC for transplantation in patients with various blood disorders. Our method utilizes a previously developed two-step culture system of megakaryopoiesis from CB CD34+ cells to generate an MK culture composed of defined MK populations: CD34+/CD41+/CD42b- MK precursors (MKP), immature CD34-/CD41+/CD42b- MK (iMK) and mature CD34-/CD41+/CD42b+ MK (mMK). While robust, the yield of MKs obtained in these cultures is restricted due to limited numbers of HSCs in CB. Our group has recently demonstrated that the numbers of CB CD34+ can be significantly expanded by epigenetic reprogramming following treatment with valproic acid (VPA). Here, we report the integration and optimization of HSC expansion with MK differentiation in order to generate a clinically relevant MK cell product. We tested 20 different culture conditions in which CD34+ cells were cultured for 5 to 8 days in the absence or presence of VPA in serum-free media with various cytokines to allow for HSC expansion. The resulting HSC pool is cultured for additional 4 to 7 days in MK differentiation/maturation media. The overall yield of CD41+ MKs obtained ranged from 8 to 33 MK per input CD34+ cell expanded in the presence of cytokines alone (n=10; mean 19.8 MK) and from 9 to 34 MK per input CD34+ cell expanded in the presence of cytokines plus VPA (n=10; mean 20.7 MK). Given that up to 2x106 CD34+ cells can be isolated from one CBU, it is anticipated that a culture yielding 28 or more MK per one CD34+ cell would generate over 56x106 MK or the equivalent of 7x105 CD41+ MK/kg/body weight for infusion into an 80 kg recipient. The culture conditions resulting in a yield of 28 or more MK per one CD34+ cell input are currently optimized to further maximize the fraction of MK generated which currently varies between 15-57% of culture. The predominant sub-population of MK resulted in these conditions consists of mMKs, regardless of VPA treatment. However, in the presence of VPA, the cultures contain a greater number of assayable CFU-MKs as compared to cytokines alone. Furthermore, preliminary studies suggest that transplantation of ex vivo generated MK leads to detectable human CD41+ cells into the BM and human PTL into the PB of NSG recipient mice. These results indicate that a MK cell product derived from CB HSCs expanded by VPA comprises not only mMK and iMK capable of immediate PTL release but also MKP and HPCs which are capable of sustained MK and PTL production. Another major advantage of a transfusion product composed of nucleated MKs is the possibility of storage by cryopreservation. Due to the fragility of mMK, we tested the cryopreservation of heterogeneous and purified MK cultures. Viability of cryopreserved MK cultures post-thaw was between 68.4-70% and no changes in the MK phenotype. Studies are ongoing to test the ex vivo and in vivo functionality of the cryopreserved MKs. In summary, starting with expanded CB HSC we created a cryopreservable cell product composed of different MK sub-populations within the MK hierarchy which is being developed into a clinically relevant therapy for treatment of thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

scholarly journals Repopulating hematopoietic stem cells from steady-state blood before and after ex vivo culture are enriched in the CD34+CD133+CXCR4low fraction

Haematologica ◽  
2018 ◽  
Vol 103 (10) ◽  
pp. 1604-1615 ◽  
Author(s):  
Véronique Lapostolle ◽  
Jean Chevaleyre ◽  
Pascale Duchez ◽  
Laura Rodriguez ◽  
Marija Vlaski-Lafarge ◽  
...  
Keyword(s):  
Stem Cells ◽  
Steady State ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Before And After ◽  
Ex Vivo Culture
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