Attenuation of Surface CXCR4 Down-Regulation in Human Cord Blood HSC during Ex Vivo Expansion Using the HUBEC Co-Culture System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1068-1068
Author(s):  
Naoko Takebe ◽  
Thomas MacVittie ◽  
Xiangfei Cheng ◽  
Ann M. Farese ◽  
Emily Welty ◽  
...  
Keyword(s):  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Receptor Internalization ◽  
Down Regulation ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Gm Csf

Abstract Down-modulation of surface CXCR4, a G-protein-coupled receptor, in hematopoietic stem cells (HSCs) undergoing ex vivo expansion culturing is considered to be one of the major causes of marrow reconstitution failure, possibly due to an HSC homing defect. Recently, it has been reported that severe combined immunodeficiency (SCID)-repopulating cells (SRC) were expanded from the CD34-enriched human adult bone marrow (ABM) or cord blood (CB) hematopoietic stem cells (HSC) using a human brain endothelial cell (HUBEC) co-culture system. We found that primitive cord blood cells expressing surface CXCR4 (82+5%) lost this capability significantly during 7 days of ex vivo expansion in the HUBEC co-culture containing the cytokines stem cell factor (SCF), flt-3, interleukin (IL)-6, IL-3, and granulocyte macrophage colony stimulating factor (GM-CSF). Expression levels of other surface proteins relevant to HSC homing, such as CD49d, CD95, CD26, or CD11a, were not down-modulated. We hypothesized that CXCR4 down-regulation was caused by a receptor internalization and tested several methods to reverse CXCR4 internalization back to the surface, such as elimination of GM-CSF in the culture media, performing a non-contact culture using the transwell, or adding either 0.3Mor 0.4M sucrose, or 25μg/ml chlorpromazine (CPZ), 24 hours prior to the analysis. CPZ and sucrose are known inhibitors of the cytokine-induced endocytosis of CXCR4 in neutrophils (Bruhl H. et al. Eur J Immunol 2003). Interestingly, 0.4M sucrose showed approximately a 2-fold increase of surface CXCR4 expression on CB CD34+ cells by flow cytometry analysis. CPZ and 0.3M sucrose showed a moderate increase expression of CXCR4. Using a transwell HUBEC co-culture system, CXCR4 surface expression on CD34+ cells was down-regulated during the ex vivo culture. In vitro HSC migration test showed 3.1-fold increase in migration compared to the control after incubation of HSC with 0.1M sucrose for 16 hours prior to the in vitro migration study. Eliminating GM-CSF from the cytokine cocktail or adding MG132 increased migration 1.36- and 1.2-fold compared to the control. We are currently performing an in vivo homing assay using nonobese diabetic (NOD)-SCID mice. In conclusion, the HUBEC ex vivo culture system down-regulates surface CXCR4 in human cord blood HSC. The mechanism of CXCR4 surface down regulation may be receptor internalization by cytokines. Sucrose may be useful in attenuation of CXCR4 surface expression in CD34+ HSC by inhibition of receptor internalization via clathrin-coated pits.

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2888-2888
Author(s):  
Ana Frias ◽  
Christopher D. Porada ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Cell Number ◽  
Human Cord Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

scholarly journals The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

Blood ◽  
2020 ◽  
Vol 136 (21) ◽  
pp. 2410-2415 ◽  
Author(s):  
Peng Hua ◽  
Joanna Hester ◽  
George Adigbli ◽  
Rong Li ◽  
Bethan Psaila ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Bet Inhibitor ◽  
Functional Studies

Abstract Although cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor–1 enhances their ability to engraft NOD/SCID mice

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3454-3457 ◽  
Author(s):  
Hanno Glimm ◽  
Patrick Tang ◽  
Ian Clark-Lewis ◽  
Christof von Kalle ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Tgf Β1

Abstract Ex vivo proliferation of hematopoietic stem cells (HSCs) is important for cellular and gene therapy but is limited by the observation that HSCs do not engraft as they transit S/G2/M. Recently identified candidate inhibitors of human HSC cycling are transforming growth factor-β1(TGF-β1) and stroma-derived factor–1 (SDF-1). To determine the ability of these factors to alter the transplantability of human HSCs proliferating in vitro, lin− cord blood cells were first cultured for 96 hours in serum-free medium containing Flt3 ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These cells were then transferred to medium containing Steel factor and thrombopoietin with or without SDF-1 and/or TGF-β1 for 48 hours. Exposure to SDF-1 but not TGF-β1 significantly increased (> 2-fold) the recovery of HSCs able to repopulate nonobese diabetic/severe combined immunodeficiency mice. These results suggest new strategies for improving the engraftment activity of HSCs stimulated to proliferate ex vivo.

Study of In Vitro Proliferation Potential of CD133 Positive Cells Derived from Umbilical Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4039-4039
Author(s):  
Ri Zhang ◽  
Wenjin Gao ◽  
Yuanyuan Sun ◽  
Jingcheng Miao ◽  
Xueguang Zhang
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Low Concentration ◽  
Tgf Β1

Abstract Transforming growth factor-beta 1 (TGF-β1) is known to maintain primitive human hematopoietic stem/progenitor cells with polyfunctional role in a quiescent state and CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. To investigate the specific effect of TGF-β1 on proliferation and differentiation of CD133 positive cells derived from umbilical cord blood (UCB) during short-term culture in vitro, CD133 positive cells from 20 fresh UCB samples were selected using Miltenyi Biotec’s CliniMACS separation device and were cultured in IMDM medium with 20% FCS in the presence of a cytokine combination of SCF, IL-6, thrombopoietin, IL-3 and Flt3-ligand for up to 2 weeks and TGF-β1 with low concentration was also added to the mediumon day 4. The proliferative response was assessed at day 7, day 10 and day 14 by evaluating the following parameters: nucleated cells (NC), clonogenic progenitors (CFU-GEMM,CFU-GM and BFU-E), and immunophenotypes (CD133 and CD34). The results showed that efficacious expansion of various hematopoietic stem/progenitor cells was constantly observed during the culture. The fold expansion of NC on day7, day10 and day14 expansion were 33.59,224.26 and 613.48, respectively. The fold expansion of CFU-GEMM, CFU-GM and BFU-E on day 10 were 24.89, 41.62 and 49.28, respectively, obviously higher than that without ex vivo expansion (P<0.05). The expansions of CD133+, CD133+CD34+ and CD34+ subpopulation on day 14 were up to 25.83-fold, 16.16-fold and 60.54-fold, respectively. Furthermore the expansion systems with TGF-β1 showed more CD133+ cells than control at every time points. Our datas suggested that the CD133+ cells from human UCB have great expansion potential for ex-vivo expansion. The low concentration of TGF-β1 may delay over-differentiation of hematopoietic stem/progenitor cells.

Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1460-1460
Author(s):  
Laura A Paganessi ◽  
Lydia Luy Tan ◽  
Sucheta Jagan ◽  
Robin Frank ◽  
Antonio M. Jimenez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Gm Csf ◽  
Cytokine Treatment ◽  
Ex Vivo Culture ◽  
Inhibitor Treatment

Abstract Abstract 1460 Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures: No relevant conflicts of interest to declare.

A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells

2005 ◽  
Vol 33 (7) ◽  
pp. 828-835 ◽  
Author(s):  
Cláudia Lobato da Silva ◽  
Raquel Gonçalves ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Culture System ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Serum Free

scholarly journals Activation of OCT4 Enhances Ex Vivo Expansion of Phenotypically Defined and Functionally Engraftable Human Cord Blood Hematopoietic Stem and Progenitor Cells By Regulating HOXB4 Expression

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4332-4332
Author(s):  
Xinxin Huang ◽  
Scott Cooper ◽  
Hal E. Broxmeyer
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Fold Increase ◽  
Lentiviral Vectors ◽  
Ex Vivo Expansion ◽  
Transcriptional Factor ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Allogeneic hematopoietic cell transplantation (HCT) is well established as a clinical means to treat patients with hematologic disorders and cancer. Human cord blood (CB) is a viable source of hematopoietic stem cells (HSC) for transplantation. However, numbers of nucleated cells retrieved, as well as limited numbers of HSC/progenitor cells (HPC) present, during collection may be problematic for treatment of adult patients with single CB HCT. One means to address the problem of limiting numbers of HSC/HPC is to ex vivo expand these cells for potential clinical use. While progress has been made in this endeavor, there is still a clinically relevant need for additional means to ex vivo expansion of human HSC and HPC. OCT4, a transcriptional factor, plays an essential role in pluripotency and somatic cell reprogramming, however, the functions of OCT4 in HSC are largely unexplored. We hypothesized that OCT4 is involved in HSC function and expansion, and thus we first evaluated the effects of OAC1 (Oct4-activating compound 1) on ex vivo culture of CB CD34+ cells in the presence of a cocktail of cytokines (SCF, TPO and Flt3L) known to ex vivo expand human HSC. We found that CB CD34+ cells treated with OAC1 for 4 days showed a significant increase (2.8 fold increase, p<0.01) above that of cytokine cocktail in the numbers of rigorously defined HSC by phenotype (Lin-CD34+CD38-CD45RA-CD90+CD49f+) and in vivo repopulating capacity in both primary (3.1 fold increase, p<0.01) and secondary (1.9 fold increase, p<0.01) recipient NSG mice. OAC1 also significantly increased numbers of granulocyte/macrophage (CFU-GM, 2.7 fold increase, p<0.01), erythroid (BFU-E, 2.2 fold increase, p<0.01), and granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM, 2.6 fold increase, p<0.01) progenitors above that of cytokine combinations as determined by colony assays. To further confirm the role of OCT4 in human HSC, we performed OCT4 overexpression in CB CD34+ cells using lentiviral vectors and found that overexpression of OCT4 also resulted in significant increase (2.6 fold increase, p<0.01) in the number of phenotypic HSC compared to control vectors. Together, our data indicate that activation of OCT4 by OAC1 or lentiviral vectors enhances ex vivo expansion of cytokine stimulated human CB HSC. HOXB4 is a homeobox transcriptional factor that appears to be an essential regulator of HSC self-renewal. Overexpression of HOXB4 results in high-level ex vivo HSC expansion. It is reported that OCT4 can bind to the promoter region of HOXB4 at the site of 2952 bp from the transcription start point. We hypothesized that activation of OCT4 might work through upregulation of HOXB4 expression to ex vivo expand HSC. We observed that the expression of HOXB4 was largely increased (2.3 fold increase, p<0.01) after culture of CB CD34+ cells with OAC1 compared to vehicle control. siRNA mediated inhibition of OCT4 resulted in the marked reduction of HOXB4 expression (p<0.01) in OAC1-treated cells indicating that OAC1 treatment lead to OCT4-mediated upregulation of HOXB4 expression in HSC. Consistently, siRNA-mediated knockdown of HOXB4 expression led to a significant reduction in the number of Lin-CD34+CD38-CD45RA-CD90+CD49f+ HSC in OAC1-treated cells (p<0.05), suggesting HOXB4 is essential for the generation of primitive HSC in OAC1-treated cells. Our study has identified the OCT4-HOXB4 axis in ex vivo expansion of human CB HSC and sheds light on the potential clinical application of using OAC1 treatment to enhance ex vivo expansion of cytokine stimulated human HSC. Disclosures Broxmeyer: CordUse: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Mesenchymal Stem Cell-Derived Microvesicles Support Ex Vivo Expansion of Cord Blood-Derived CD34+Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Hui Xie ◽  
Li Sun ◽  
Liming Zhang ◽  
Teng Liu ◽  
Li Chen ◽  
...  
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells

Mesenchymal stem cells (MSCs) are known to support the characteristic properties of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow hematopoietic microenvironment. MSCs are used in coculture systems as a feeder layer for the ex vivo expansion of umbilical cord blood (CB) to increase the relatively low number of HSPCs in CB. Findings increasingly suggest that MSC-derived microvesicles (MSC-MVs) play an important role in the biological functions of their parent cells. We speculate that MSC-MVs may recapitulate the hematopoiesis-supporting effects of their parent cells. In the current study, we found MSC-MVs containing microRNAs that are involved in the regulation of hematopoiesis. We also demonstrated that MSC-MVs could improve the expansion of CB-derived mononuclear cells and CD34+cells and generate a greater number of primitive progenitor cells in vitro. Additionally, when MSC-MVs were added to the CB-MSC coculture system, they could improve the hematopoiesis-supporting effects of MSCs. These findings highlight the role of MSC-MVs in the ex vivo expansion of CB, which may offer a promising therapeutic approach in CB transplantation.

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