Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1097-1105 ◽  
Author(s):  
G. Güenechea ◽  
J.C. Segovia ◽  
B. Albella ◽  
M. Lamana ◽  
M. Ramı́rez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P < .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

Delayed Engraftment of Nonobese Diabetic/Severe Combined Immunodeficient Mice Transplanted With Ex Vivo–Expanded Human CD34+ Cord Blood Cells

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 1097-1105 ◽  
Author(s):  
G. Güenechea ◽  
J.C. Segovia ◽  
B. Albella ◽  
M. Lamana ◽  
M. Ramı́rez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract The ex vivo expansion of hematopoietic progenitors is a promising approach for accelerating the engraftment of recipients, particularly when cord blood (CB) is used as a source of hematopoietic graft. With the aim of defining the in vivo repopulating properties of ex vivo–expanded CB cells, purified CD34+ cells were subjected to ex vivo expansion, and equivalent proportions of fresh and ex vivo–expanded samples were transplanted into irradiated nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. At periodic intervals after transplantation, femoral bone marrow (BM) samples were obtained from NOD/SCID recipients and the kinetics of engraftment evaluated individually. The transplantation of fresh CD34+ cells generated a dose-dependent engraftment of recipients, which was evident in all of the posttransplantation times analyzed (15 to 120 days). When compared with fresh CB, samples stimulated for 6 days with interleukin-3 (IL-3)/IL-6/stem cell factor (SCF) contained increased numbers of hematopoietic progenitors (20-fold increase in colony-forming unit granulocyte-macrophage [CFU-GM]). However, a significant impairment in the short-term repopulation of recipients was associated with the transplantation of the ex vivo–expanded versus the fresh CB cells (CD45+repopulation in NOD/SCIDs BM: 3.7% ± 1.2% v 26.2% ± 5.9%, respectively, at 20 days posttransplantation; P &lt; .005). An impaired short-term engraftment was also observed in mice transplanted with CB cells incubated with IL-11/SCF/FLT-3 ligand (3.5% ± 1.7% of CD45+ cells in femoral BM at 20 days posttransplantation). In contrast to these data, a similar repopulation with the fresh and the ex vivo–expanded cells was observed at later stages posttransplantation. At 120 days, the repopulation of CD45+ and CD45+/CD34+ cells in the femoral BM of recipients ranged between 67.2% to 81.1% and 8.6% to 12.6%, respectively, and no significant differences of engraftment between recipients transplanted with fresh and the ex vivo–expanded samples were found. The analysis of the engrafted CD45+ cells showed that both the fresh and the in vitro–incubated samples were capable of lymphomyeloid reconstitution. Our results suggest that although the ex vivo expansion of CB cells preserves the long-term repopulating ability of the sample, an unexpected delay of engraftment is associated with the transplantation of these manipulated cells.

Kinetics of Engraftment and Graft Versus Host Disease After Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units In Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3722-3722
Author(s):  
Li Ming Ong ◽  
Xiubo Fan ◽  
Pak Yan Chu ◽  
Florence Gay ◽  
Justina Ang ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Insulin Growth Factor ◽  
Nsg Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Abstract 3722 Ex vivo expansion of cord blood (CB) hematopoietic stem cells (HSCs) and cotransplantation of two CB units can enhance applicability of CB transplants to adult patients. This is the first study on cotransplantation of ex vivo expanded and unexpanded human CB units in immunodeficient mice, simulating conditions for ex vivo CB expansion clinical trials. CB units were cultured in serum-free medium supplemented with Stem Cell Factor, Flt-3 ligand, Thrombopoietin and Insulin Growth Factor Binding Protein-2 with mesenchymal stromal co-culture. Cotransplantation of unexpanded and expanded CB cells was achieved by tail vein injection into forty-five sublethally irradiated nonobese diabetic SCID-IL2γ−/− (NSG) mice. Submandibular bleeding was performed monthly and mice were sacrificed 4 months following transplantation to analyze for human hematopoietic engraftment. CB expansion yielded 40-fold expansion of CD34+ cells and 18-fold expansion of HSCs based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts had 4.30% human cell repopulation, compared to 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses (p = 0.07). Ex vivo expanded grafts with lower initiating cell doses also had equivalent engraftment to unexpanded grafts with higher cell dose (8.0% vs 7.9%, p= 0.93). However, the unexpanded graft, richer in T-cells, predominated in final donor chimerism. Ex vivo expansion resulted in enhanced CB engraftment at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis. The expanded graft with increased stem/progenitor cells enhanced initial engraftment despite eventual rejection by the unexpanded graft. Disclosures: No relevant conflicts of interest to declare.

Ex Vivo Expansion of Frozen Cord Blood Products without Selection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2844-2844
Author(s):  
Ian K. McNiece ◽  
Jenny Harrington ◽  
Joshua Kellner ◽  
Jennifer Turney ◽  
Elizabeth J. Shpall
Keyword(s):  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
In Vivo Studies ◽  
Adherent Cells ◽  
Blood Products ◽  
Cd34 Cells ◽  
Duke University

Abstract Ex vivo expansion of cord blood products (CB) has been proposed as an approach to increase the number of cells available from a single CB unit. We and others have reported the requirement of CD34 selection for optimal expansion of CB products, however, the selection of frozen CB products results in significant losses of CD34+ cells with a median recovery of 43% (range 6 to 203%, N=40) and low purities resulting in decreased expansion. Therefore we explored approaches to expand CB without prior selection and have described the use of co-culture of CB mononuclear cells (MNC) on mesenchymal stem cells (MSC). In the present study we have evaluated the expansion of clinical CB products (provided by Duke University CB Bank CB). MNC were obtained after ficol separation of RBCs and 10% of the CB product was cultured on preformed layers of MSC in T150 flasks containing 50ml of defined media (Sigma Aldrich) plus 100 ng/ml each of rhSCF, rhG-CSF and rhTpo. After 6 days of culture, the non adherent cells were transferred to a Teflon bag and a further 50 ml of media and GFs added to the flask. Again at day 10, non adherent cells were transferred to the Teflon bag and media and growth factors replaced. At day 12 to 13 of incubation the cells were harvested, washed and total nucleated cell (TNC) counts and progenitor assays performed. In three separate experiments we have achieved greater than 20 fold expansion of TNC with a median of 22, and a median expansion of GM-CFC of 37 fold. Morphologic analysis demonstrated the expanded cells contained high levels of mature neutrophils and neutrophil precursors. In vivo studies in NOD/SCID mice also demonstrated that the expanded cells maintained in vivo engraftment potential. Clinical studies are being designed to evaluate the in vivo potential of CB MNC products expanded on MSC.

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.

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.

scholarly journals In Vivo Analysis of PML-RARA in a Humanized Mouse Model

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1020-1020
Author(s):  
Hiromichi Matsushita ◽  
Takashi Yahata ◽  
Yin Sheng ◽  
Yoshihiko Nakamura ◽  
Yukari Muguruma ◽  
...  
Keyword(s):  
Cord Blood ◽  
Conflicts Of Interest ◽  
Nuclear Bodies ◽  
Leukemic Cells ◽  
Human Cord Blood ◽  
Immunodeficient Mice ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. Disclosures No relevant conflicts of interest to declare.

Ectopic Expression of Mutated HOXB4 Proteins with Increased Intracellular Stability Affects Both Long and Short Term Hematopoietic Reconstitution.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3183-3183
Author(s):  
Stephanie Beauchemin ◽  
Gorazd Krosl ◽  
Nathalie Beslu ◽  
Jana Krosl ◽  
Guy Sauvageau ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Ex Vivo ◽  
Ectopic Expression ◽  
Ex Vivo Expansion ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Post Transplantation ◽  
Blood Leukocytes

Abstract The capacity of recombinant Hoxb4 protein to induce ex vivo expansion of HSCs identifies this protein as a potential HSC expanding factor. However, its short extra- and intra-cellular half-life (3–4 hours and 40–60 minutes, respectively) are hampering clinical applications of Hoxb4. The analyses of Hoxb4 molecular structure lead us to generate amino acid substitutions: Leu7→Ala, Tyr23→Ala and Tyr28→Ala in the Hoxb4 protein in order to decrease its degradation. Indeed, these modifications increased the intracellular stability of Hoxb4 protein ~3-fold compared to wild type Hoxb4 (Hoxb4(WT)). The ability of mutated Hoxb4 protein to favor expansion of hematopoietic progenitors was first examined in cultures initiated with 10% Hoxb4(WT)-GFP, 10% mutated Hoxb4-YFP expressing cells and 80% non-transduced cells. After an 18-day culture, the proportion of Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) cells increased to 50–60% in comparison to 30% for Hoxb4(WT) (p < 0.05), and no difference between the proliferation of Hoxb4(Tyr28→Ala) and Hoxb4(WT) cells could be identified. Western blot analyses showed that these Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) cells expressed ~ 4-fold higher and Hoxb4(Tyr28→Ala) cells ~ 8-fold lower levels of Hoxb4 protein than Hoxb4(WT) cells. The long-term reconstituting ability of these constructs was then evaluated in vivo using competitive repopulation assays. At 8 weeks after transplantation, Hoxb4(Leu7→Ala) and Hoxb4(Tyr23→Ala) contributed to 11.5±2 and 13.1±1.8% of peripheral blood leukocytes (PBL) compared to 26.2± 4.3% determined for Hoxb4(WT), while after 16 weeks the progeny of Hoxb4(WT) cells generated the majority (≥65%) of the transplant-derived PBL in all recipients. Likewise, 16 weeks post transplantation Hoxb4 positive cells represented ≥80% of bone marrow, while cells expressing mutated Hoxb4 were present at ~10–12%level. Flow cytometry analysis of bone marrow, spleen and thymus revealed that mutated Hoxb4, like Hoxb4(WT) was expressed by all hematopoietic lineages, and that repopulation differences observed between mutated and WT Hoxb4 expressing cells were almost entirely attributable to myeloid lineage cells. However, short-term, non-competitive repopulation experiments showed that in the first 4 weeks post transplantation, mutated Hoxb4 expressing progenitors had a significantly greater contribution to the PBL recovery in comparison to Hoxb4(WT) (range 50–70% vs 16–30%, respectively; p < 0.05) for all three mutant proteins. Interestingly, this difference became less pronounced and non-significant after week 8 post transplantation. Together, these studies strongly suggest that different intracellular levels of Hoxb4 protein are affecting different types of hematopoietic progenitors. Early ex vivo expansion of clonogenic progenitors was achieved with mutated Hoxb4 proteins without impairing HSC long-term reconstituting ability. Thus, mutated Hoxb4 could represent a useful tool to accelerate engraftment after HSC transplantation.

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Blood ◽  
2009 ◽  
Vol 114 (24) ◽  
pp. 5044-5051 ◽  
Author(s):  
Isabelle I. Salles ◽  
Tim Thijs ◽  
Christine Brunaud ◽  
Simon F. De Meyer ◽  
Johan Thys ◽  
...  
Keyword(s):  
Cord Blood ◽  
Flow Model ◽  
Ex Vivo ◽  
Human Platelets ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Nonobese Diabetic ◽  
Cd34 Cells ◽  
Cord Blood Cd34

Abstract Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34+ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 × 103/μL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ibα (GPIbα) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

scholarly journals SALL4 Is a Key Factor in HDAC Inhibitor Mediated Ex Vivo Expansion of Human Peripheral Blood Mobilized Stem/Progenitor CD34+CD90+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1566-1566 ◽  
Author(s):  
Hiro Tatetsu ◽  
Fei Wang ◽  
Chong Gao ◽  
Shikiko Ueno ◽  
Xi Tian ◽  
...  
Keyword(s):  
Cord Blood ◽  
Peripheral Blood ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cd34 Cells

Abstract Hematopoietic stem cells (HSCs) possess the unique capacity to self-renew and give rise to all types of mature cells within the blood and immune systems. Despite our progress in understanding the molecular factors that support the self-renewal and differentiation of the hematopoietic system in vivo, less is known on how to modulate the factors that govern the self-renewal of hematopoietic stem/progenitor cells (HSPCs) ex vivo. Unlike in the case of embryonic stem (ES) cells, expansion of CD34+ HSPC in culture in general is at the expense of loss of “stemness”. HSPCs can be collected from cord blood (CB), mobilized peripheral blood (PBSC), and rarely bone marrow (BM) at the present practice. Due to the limited CD34+ cell number in one single cord blood unit, much of the current efforts on developing technology of ex vivo expansion of HSPC uses cord blood as a source and is clinically applied to cord blood HSPC transplants. However, there are growing needs for expanding PBSCs for transplant-related practices such as HSPC expansion from poor autologous mobilizations, gene therapy or genome-editing via TALENs or CRISPR/Cas9. Developing a technology that would allow HSPC ex vivo expansion from both CB and PBSC sources is a key step towards this goal. Several groups have reported that ex vivo culture of CB CD34+ cells with HDAC inhibitors (HDACi) can lead to expansion of a CD34+CD90+ population, which is responsible for enhanced marrow-repopulating potential. In this study, we ask whether HDACi can have a similar effect on PBSC CD34+ cells. Furthermore, we have explored the mechanism(s) mediated by HDACi in CD34+CD90+ cell expansion. First we assessed a panel of HDACi to identify the most potent molecule for CD34+CD90+ cell expansion and selected trichostatin A (TSA) for future study. Next, TSA was added to the cytokines (SCF, Flt3 ligand, IL-3 and IL-6) to further characterize its potential in PBSC CD34+CD90+ cell expansion. We observed TSA treated CD34+ cultures with cytokines yielded 4.8 times greater numbers of CD34+CD90+ cells as compared to the cultures containing cytokines with DMSO solvent control. We next examined SCID repopulating ability (SRA) to evaluate the cultured CD34+CD90+ cells in vivo. We observed that mice transplanted with 3 million CD34+ cells treated with TSA had higher degree of human cell chimerism than those treated with DMSO at 8 weeks bone marrow and peripheral blood (32% vs 18%; p < 0.05), which was further confirmed by secondary transplantation. Furthermore, these cells were capable of differentiating into cells belonging to multiple hematopoietic lineages. To investigate the molecular mechanisms responsible for the expansion of functional HSCs and progenitors that were observed following TSA treatment, we analyzed the expression levels of several HSPC related genes, which were compared between CD34+ cells treated with TSA and DMSO. Significantly higher transcript levels were detected for GATA 2 (p < 0.05), HOXB4 (p < 0.05), HOXA9 (p < 0.05), and SALL4 (p < 0.05) by real time quantitative RT-PCR in TSA expanded cells as compared with controls. To evaluate whether these transcription factors can contribute to the expansion of CD34+CD90+ cells, GATA2, HOXB4 or SALL4 shRNAs were transfected into PBSC CD34+ cells, followed by culture with TSA. Among these transcription factors, knocking down SALL4 expression led to the most significant reduction of CD34+CD90+ cell numbers (33% of reduction). In addition, overexpression of SALL4 in PBSC CD34+ cells led to an increase of CD34+CD90+ cell numbers when compared to controls (p < 0.05). Overall, our study demonstrated a novel HDACi mediated ex vivo PBSC culture technology that leads to the expansion of CD34+CD90+ cells and an increase of the marrow repopulating potential of these cells. Both gain-of-function and loss-of-function studies support that SALL4 is a key transcription factor responsible for the process. Future study on the use of HDACi or other methods to increase SALL4 expression/function will be highly beneficial to ex vivo HSPC (CB and PBSC) expansion technology. Disclosures No relevant conflicts of interest to declare.

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