Ex Vivo Expansion of Human SCID-Repopulating Cells Using Recombinant TAT-HOXB4 Protein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3159-3159 ◽  
Author(s):  
Gorazd Krosl ◽  
Marie-Pier Giard ◽  
Jana Krosl ◽  
Stephanie Beauchemin ◽  
Keith Humphries ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Ex Vivo ◽  
Day Treatment ◽  
Fold Increase ◽  
Limited Information ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Short Term Exposure

Abstract One of the major obstacles to the successful clinical application of hematopoietic stem cell (HSC) transplantation, particularly in the context of related haplotype-mismatched transplantation, unrelated cord blood transplants for adults, and grafts that are processed ex vivo to remove malignant, or alloreactive T cells, is the number of available long-term repopulating HSCs. The addition of soluble recombinant TAT-HOXB4 protein was recently reported to enable rapid in vitro expansion of murine HSCs that retain their in vivo proliferation and differentiation capacity. However, the ability of this recombinant protein to effectively expand human hematopoietic stem cells remains hypothetical. In addition, limited information is available on underlying mechanisms of HOXB4 HSC expansion. First, to determine the capacity of recombinant TAT-HOXB4 protein to promote human HSC expansion, we treated human CD34+ cells for 4 and 8 days with 40 nM, or 80 nM TAT-HOXB4 protein in X-Vivo 15 medium supplemented with Stem Cell Factor, TPO, IL-6 and Flt3-ligand. Cultures exposed to TAT-HOXB4 treatment for 8 days had no pronounced effect on the total cell yield. During this period, a 2-fold net loss of CFU-GEMM was observed for controls, in comparison to ~8-fold and ~5-fold expansions in response to 40 nM and 80 nM TAT-HOXB4 (p<0.05), respectively. Recombinant TAT-HOXB4 also induced ~10–15-fold expansion of large CFU-GM, compared to only ~2.5-fold increase for controls (p<0.05). HSC numbers were enumerated at the beginning and at the end of the treatment using the principle of limiting dilution in a 4-month NOD/SCID repopulation assay. Culture for 8 days in cytokines devoid of TAT-HOXB4 resulted in ~2-fold loss of SCID Repopulating Cells (SRCs), while cultures supplemented with 40 and 80 nM TAT-HOXB4 protein showed a 2.5-fold (95% CI 1.7 – 3.3 fold) and 5.5-fold (95% CI 3.6 – 7.4 fold) increase, respectively. Then, to determine whether the increase in HSC numbers resulted from HOXB4-enhanced proliferation of HSCs, we examined the cell cycle profile of control and HOXB4-treated cell populations using Hoechst 33342 and pyronin Y dyes. After 4-day treatment with 80 nM TAT-HOXB4, 44%±12% of CD34+CD38+ cells were in Go, compared to only 19%±6% of the controls (p<0.05). In contrast, similar proportions (89–91%) of quiescent CD34+CD38− cells were observed for both conditions. Tracking cell divisions using CFSE also showed that during this period, HOXB4-treated CD34+CD38+ cells underwent ~2 population doublings less than controls (p<0.05). In conclusion, short-term exposure of human CD34+ populations to recombinant TAT-HOXB4 protein has the potential to achieve clinically relevant HSC expansion levels. At the HSC level, these observations suggest that TAT-HOXB4 preferentially affects cell fate (self-renewal?) rather than cell proliferation.

Screen for Small Molecules Capable of Expanding Human Hematopoietic Stem Cell Ex Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1919-1919
Author(s):  
Iman Hatem Fares ◽  
Jalila Chagraoui ◽  
Jana Krosl ◽  
Denis-Claude Roy ◽  
Sandra Cohen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 1919 Hematopoietic stem cell (HSC) transplantation is a life saving procedure whose applicability is restricted by the lack of suitable donors, by poor responsiveness to mobilization regimens in preparation of autologous transplantations, by insufficient HSC numbers in individual cord blood units, and by the inability to sufficiently amplify HSCs ex vivo. Characterization of Stemregenin (SR1), an aryl hydrocarbon receptor (AHR) antagonist that promotes HSC expansion, provided a proof of principle that low molecular weight (LMW) compounds have the ability to promote HSC expansion. To identify novel putative agonists of HSC self-renewal, we initiated a high throughput screen (HTS) of a library comprising more than 5,000 LMW molecules using the in vitro maintenance of the CD34+CD45RA- phenotype as a model system. Our study was based on the fact that mobilized peripheral blood-derived CD34+CD45RA- cells cultured in media supplemented with: stem cell factor, thrombopoietin, FLT3 ligand and interleukin 6, would promote the expansion of mononuclear cells (MNC) concomitant with a decrease in CD34+CD45RA- population and HSC depletion. LMW compounds preventing this loss could therefore act as agonists of HSC expansion. In a 384-well plate, 2000 CD34+cells were initially cultured/well in 50μl medium comprising 1μM test compounds or 0.1% DMSO (vehicle). The proportions of CD34+CD45RA− cells were determined at the initiation of experiment and after a 7-day incubation. Six of 5,280 LMW compounds (0.11%) promoted CD34+CD45RA− cell expansion, and seventeen (0.32%) enhanced differentiation as determined by the increase in proportions of CD34−CD45RA+ cells compared to control (DMSO). The 6 LMW compounds promoting expansion of the CD34+CD45RA− cell population were re-analyzed in a secondary screen. Four out of these 6 molecules suppressed the transcriptional activity of AHR, suggesting that these compounds share the same molecular pathway as SR1 in stimulating HSC expansion, thus they were not further characterized. The remaining 2 compounds promoted, similar to SR1 or better, a 10-fold and 35-fold expansion of MNC during 7 and 12-day incubations, respectively. The expanded cell populations comprised 65–75% of CD34+ cells compared to 12–30% determined for DMSO controls. During 12-day incubation with these compounds, the numbers of CD34+ cells increased ∼25-fold over their input values, or ∼ 6-fold above the values determined for controls. This expansion of CD34+ cells was associated with a ∼5-fold increase in the numbers of multilineage CFC (granulocyte, erythroid, monocyte, and megakaryocyte, or CFU-GEMM) compared to that found in DMSO control cultures. The ability of the 2 newly identified compounds to expand functional HSCs is currently being evaluated in vivo usingimmunocompromised mice. In conclusion, results of our initial screen suggest that other mechanism, besides inhibition of AhR, are at play for expansion of human HSC. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Histone Deacetylase Inhibitors As Enhancers of Human Hematopoietic Stem Cell Activity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5044-5044
Author(s):  
Manja Wobus ◽  
Guruchandar Arulmozhivarman ◽  
Martin Kraeter ◽  
Jens Friedrichs ◽  
Martin Stoeter ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Hdac Inhibitors ◽  
Cell Activity ◽  
Adhesion Assay ◽  
Hematopoietic Stem ◽  
Notch 3 ◽  
Human Cd34 ◽  
Stem Cell Activity

Abstract Introduction The identification of compounds which increase the number but also keep or enhance the activity of hematopoietic stem and progenitor cells (HSPCs) could improve the clinical outcome after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). So far, most attempts to increase HSPC numbers ex vivo have been unsuccessful because of either inadequate cell numbers and/or loss of engraftment capacity and HSPC quality during expansion. Executing drug discovery screens in vertebrate systems is generally expensive, technically challenging and time consuming. Therefore, the zebrafish represents a versatile vertebrate model allowing HSPC regulation and development studies during embryogenesis and adulthood. Methods We used a semi-automated chemical screen to identify modulators of HSPC activity by transgenic (cmyb:EGFP) zebrafish embryos. Verification of identified histone deacetylase (HDAC) inhibitor candidates was carried out in vitro using human CD34+ HSPCs which were isolated from apharesis samples of healthy donors after mobilization with G-CSF by anti-CD34 coupled magnetic beads. The influence of HDAC inhibitors on HSPC phenotype, gene expression pattern as well as adhesion and migration capacity was analyzed after 5 days of treatment either in single or in co-culture with bone marrow-derived mesenchymal stromal cells (MSCs). Results The HDAC inhibitors valproic acid (VPA), resminostat and entinostat were shown to significantly amplify the number of hematopoietic precursors in a chemical in vivo zebrafish embryo screen (Arulmozhivarman et al. 2016). Treatment of human CD34+ HSPCs with these compounds in vitro resulted in a significantly increased percentage of CD34+CD90+ cells up to 60% compared to controls which showed only 2% of double positive cells as well as in 3-fold higher CD34+ and about 12-fold higher CD34+CD90+ absolute cell numbers. CD34 is a well-known surface marker for human immature HSPCs and in combination with CD90 it defines a potentially pluripotent subpopulation. In a co-culture setting, we found that VPA treated cells showed 2 to 3-fold higher attachment capacity on MSCs compared to the control cells. This finding led us to quantify the adhesive capacity of cells using static adhesion assay and atomic force microscopy based single-cell force spectroscopy (AFM-SCFS). Interestingly, detachment forces of VPA treated HSPCs were 3 times increased on MSCs compared to control cells and a similar phenotype was observed by static adhesion assay. Accordingly, the chemokine-mediated migration of VPA treated HSPCs towards SDF-1/CXCL12 was inhibited. To reveal underlying downstream molecules and mechanisms mediating the modified cellular characteristics, a whole genome expression array was carried out for HSPCs treated with VPA in comparison to untreated controls. Amongst a panel of regulated genes, the melanoma cell adhesion molecule (MCAM/CD146), Notch 3 and its downstream effector Hes-1 as well as the SDF-1 receptor CXCR-4 were found to be significantly changed. Whereas the decreased expression of CXCR4 correlates with the inhibited migration potential of VPA-treated HSPCs and Notch-3/Hes-1 have a known role in normal and malignant hematopoiesis (Gu et al. 2016), the induced expression of MCAM on HSPCs was not described so far. The result was confirmed by flow cytometry which revealed a 40% MCAM-positive cell population when treated with VPA, whereas the control showed only negative cells. Additionally, significant higher transcript levels were detected for MCAM by quantitative real-time PCR in VPA expanded cells. Recently, we described a role of MCAM in MSCs for the hematopoietic support (Stopp et al. 2013). The inducible expression in HSPCs may reflect homotypic interactions which preserve a more immature subpopulation with high stem cell activity. Conclusion We describe for the first time the ability of the HDAC inhibitors VPA, resminostat and entinostat to efficiently expand CD34+ HSPCs ex vivo especially supporting a CD34+CD90+ subpopulation with potentially high stem cell activity. Moreover, a potential role of MCAM in this context may offer new perspectives of the HSPC expansion ex vivo for the improvement of HSCT. Disclosures No relevant conflicts of interest to declare.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

Maintaining the self-renewal and differentiation potential of human CD34+ hematopoietic cells using a single genetic element

Blood ◽  
2003 ◽  
Vol 102 (13) ◽  
pp. 4369-4376 ◽  
Author(s):  
James C. Mulloy ◽  
Jorg Cammenga ◽  
Francisco J. Berguido ◽  
Kaida Wu ◽  
Ping Zhou ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Hematopoietic Cells ◽  
The Self ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Human Cd34

AbstractHematopoiesis is a complex process involving hematopoietic stem cell (HSC) self-renewal and lineage commitment decisions that must continue throughout life. Establishing a reproducible technique that allows for the long-term ex vivo expansion of human HSCs and maintains self-renewal and multipotential differentiation will allow us to better understand these processes, and we report the ability of the leukemia-associated AML1-ETO fusion protein to establish such a system. AML1-ETO-transduced human CD34+ hematopoietic cells routinely proliferate in liquid culture for more than 7 months, remain cytokine dependent for survival and proliferation, and demonstrate self-renewal of immature cells that retain both lymphoid and myeloid potential in vitro. These cells continue to express the CD34 cell surface marker and have ongoing telomerase activity with maintenance of telomere ends, however they do not cause leukemia in nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. Identification of the signaling pathways that are modulated by AML1-ETO and lead to the self-renewal of immature human progenitor cells may assist in identifying compounds that can efficiently expand human stem and progenitor cells ex vivo. (Blood. 2003; 102:4369-4376)

Pre-Transplant Integration Site Diagnostics in Hematopoietic Stem Cell Gene Transfer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3581-3581
Author(s):  
Claudia R Ball ◽  
Sylvia Fessler ◽  
Daniela Belle ◽  
Manfred Schmidt ◽  
Christof von Kalle ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Cell Clones ◽  
Integration Sites ◽  
Cell Gene ◽  
Stem Cell Gene

Abstract Abstract 3581 Poster Board III-518 We and others have previously shown that insertional activation of cellular genes caused by integrated retroviral vectors can lead to clonal dominance and malignant transformation. Pre-transplant diagnostics of vector flanking sequences and subsequent elimination of those clones that carry potentially dangerous integration sites prior to transplantation would dramatically improve the safety of clinical gene therapy regimens. Such a strategy requires efficient transduction of few or individual stem cells, their in vitro amplification and highly sensitive integration site determination before transplantation. To define optimal time points for transduction and ascertain the transplantability of ex vivo expanded murine stem cell clones, single CD45+Lin−Rho+SP cells isolated from bone marrow of male C57BL/6J (B6J) mice were cultivated for 8-10 days in the presence of IL11, SCF and Flt3-L. 10% of the sorted cells formed clones in vitro. In 28% ± 5% of these clones, the first division occurred during the first 48 hours after sorting, another 32% ± 8% divided up to 72 hours after sorting and additional 33% ± 7% up to 96 hours after sorting. 7% ± 4% had undergone their first division at a later time point. To examine the transplantability after ex vivo expansion, individual cell clones (containing 12 to >600 cells) were transplanted together with 105 carrier cells into lethally irradiated sex-mismatched syngeneic mice. The presence of donor-derived cells in peripheral blood of 20 transplanted mice was analyzed by Y-chromosome specific PCR. 55% of the ex vivo expanded clones contributed to post-transplant hematopoiesis. 25% of these clones exhibited long-term activity for >6 months after transplantation. Interestingly, only cell clones that had undergone their first division 48-96 hours after cell sorting contributed to long-term post-transplant hematopoiesis. For transduction, individual stem cell clones were spinoculated for 60 minutes with a GFP encoding lentiviral vector (MOI 100-5000). 5 days after transduction, 50% of cells generated by each clone were harvested, lysed and analyzed by LAM-PCR and integration site sequencing. After an additional 3 days, single clones were transplanted together with 105 carrier cells into lethally irradiated congeneic B6.SJL-PtprcaPepcb/BoyJ mice. Four weeks after transplantation, in 30% of these mice ≥0.4% CD45.1+ cells derived from single cell clones were detected in the peripheral blood. In 50% of these mice, the transduced clones contributed to myelopoiesis as well as lymphopoiesis for more than 24 weeks after transplantation, demonstrating that the longterm hematopoietic stem cell potential was retained after single cell marking and expansion. These results demonstrate that single stem cell gene transfer and subsequent expansion is possible to allow integration site determination. Long-term stem cells with defined lentiviral integration sites can be selected for transplantation. In summary, we provide proof of concept that pre-transplant diagnostics of integration sites is feasible to increase the safety of gene therapy by eliminating stem cell clones from transplants that carry unwanted integration sites. Disclosures: No relevant conflicts of interest to declare.

Recombinant DEK Enhances Ex Vivo expansion of Human Cord Blood and Mouse Bone Marrow Hematopoietic Stem Cells in a CXCR2- and Hspg-Dependent Manner

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 779-779
Author(s):  
Maegan L. Capitano ◽  
Nirit Mor-Vaknin ◽  
Maureen Legendre ◽  
Scott Cooper ◽  
David Markovitz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Colony Formation ◽  
Ex Vivo ◽  
Fold Increase ◽  
Inhibitory Effect ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Inhibitory Function

Abstract DEK is a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin remodeling, and mRNA processing. Endogenous DEK regulates hematopoiesis, as BM from DEK-/- mice manifest increased hematopoietic progenitor cell (HPC) numbers and cycling status and decreased long-term and secondary hematopoietic stem cell (HSC) engrafting capability (Broxmeyer et al., 2012, Stem Cells Dev., 21: 1449; 2013, Stem Cells, 31: 1447). Moreover, recombinant mouse (rm) DEK inhibits HPC colony formation in vitro. We now show that rmDEK is myelosuppressive in vitro in an S-phase specific manner and reversibly decreases numbers (~2 fold) and cycling status of CFU-GM, BFU-E, and CFU-GEMM in vivo, with DEK-/- mice being more sensitive than control mice to this suppression. In contrast, in vivo administration of rmDEK to wild type and DEK-/- mice enhanced numbers of phenotypic LT-HSC. This suggests that DEK may enhance HSC numbers by blocking production of HPCs. We thus assessed effects of DEK on ex vivo expansion of human CD34+ cord blood (CB) and mouse Lin- BM cells stimulated with SCF, Flt3 ligand, and TPO. DEK significantly enhanced ex vivo expansion of rigorously-defined HSC by ~3 fold both on day 4 (~15 fold increase from day 0) and 7 (~29 fold increase from day 0) when compared to cells expanded without DEK. Expanding HSC with DEK also resulted in a decrease in the percentage of apoptotic HSC. Further studies were done to better define how DEK works on HSC and HPC. As extracellular DEK can bind to heparan sulfate proteoglycans (HSPG), become internalized, and then remodel chromatin in non-hematopoietic cells in vitro (Kappes et al., 2011, Genes Dev., 673; Saha et al., 2013, PNAS, 110: 6847), we assessed effects of DEK on the heterochromatin marker H3K9He3 in the nucleus of purified mouse lineage negative, Sca-1 positive, c-Kit positive (LSK) BM cells by imaging flow cytometry. DEK enhanced the presence of H3K9Me3 in the nucleus of DEK-/- LSK cells, indicating that rmDEK can be internalized by LSK cells and mediate heterochromatin formation. We also investigated whether inhibiting DEK's ability to bind to HSPG would block the inhibitory function of DEK in HPC. Blocking the synthesis of, the surface expression of, and the binding capability of HSPG blocked the inhibitory effect of DEK on colony formation. Blocking the ability of DEK to bind to HSPG also blocks the expansion of HSC in ex vivo expansion assays, suggesting that DEK mediates its function in both HSC and HPC by binding to HSPG but with opposing effects. To further evaluate the biological role of rmDEK, we utilized single-stranded anti-DEK aptamers that inactivate its function. These aptamers, but not their control, neutralized the inhibitory effect of rmDEK on HPC colony formation. Moreover, treating BM cells in vitro with truncated rmDEK created by incubating DEK with the enzyme DPP4 (DEK has targeted truncation sites for DPP4) eliminated the inhibitory effects of DEK, suggesting that DEK must be in its full- length form in order to perform its function. Upon finding that DEK has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, and as DEK is a chemoattractant for mature white blood cells, we hypothesized that DEK may manifest at least some of its actions through CXCR2, the receptor known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case, we first confirmed expression of CXCR2 on the surface of HSC and HPC and then determined if neutralizing CXCR2 could block DEK's inhibitory function in HPC. BM treated in vitro with rmDEK, rhIL-8, or rmMIP-2 inhibited colony formation; pretreating BM with neutralizing CXCR2 antibodies blocked the inhibitory effect of these proteins. DEK inhibition of CFU-GM colony formation is dependent on Gai-protein-coupled receptor signaling as determined through the use of pertussis toxin, which is a mechanism unique to DEK, as we have previously reported that IL-8 and MIP-1a are insensitive to the inhibitory effects of pertussis toxin. Blocking the ability of DEK to bind to CXCR2 also inhibited the expansion of HSC in an ex vivo expansion assay. This suggests that DEK binds to CXCR2, HSPG or both to mediate its function on HPC and HSC, enhancing HSC but decreasing HPC numbers. Therefore, DEK may be a crucial regulatory determinant of HSC/HPC function and fate decision that is utilized to enhance ex vivo expansion of HSC. Disclosures No relevant conflicts of interest to declare.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2244-2252 ◽  
Author(s):  
Jean-François Arrighi ◽  
Conrad Hauser ◽  
Bernard Chapuis ◽  
Rudolf H. Zubler ◽  
Vincent Kindler
Keyword(s):  
Stem Cell ◽  
Stem Cell Factor ◽  
Fold Increase ◽  
Cell Number ◽  
Interleukin 4 ◽  
Flt3 Ligand ◽  
Gm Csf ◽  
Human Cd34 ◽  
Cd34 Cells

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Enforced expression of cyclin D2 enhances the proliferative potential of myeloid progenitors, accelerates in vivo myeloid reconstitution, and promotes rescue of mice from lethal myeloablation

Blood ◽  
2004 ◽  
Vol 104 (4) ◽  
pp. 986-992 ◽  
Author(s):  
Yutaka Sasaki ◽  
Christina T. Jensen ◽  
Stefan Karlsson ◽  
Sten Eirik W. Jacobsen
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Proliferative Potential ◽  
Cyclin D2 ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Myeloid Progenitors

AbstractSevere and prolonged cytopenias represent a considerable problem in clinical stem cell transplantations. Cytokine-induced ex vivo expansion of hematopoietic stem and progenitor cells has been intensively explored as a means of accelerating hematopoietic recovery following transplantation but have so far had limited success. Herein, overexpression of D-type cyclins, promoting G0/G1 to S transition, was investigated as an alternative approach to accelerate myeloid reconstitution following stem cell transplantation. With the use of retroviral-mediated gene transfer, cyclin D2 was overexpressed in murine bone marrow progenitor cells, which at limited doses showed enhanced ability to rescue lethally ablated recipients. Competitive repopulation studies demonstrated that overexpression of cyclin D2 accelerated myeloid reconstitution following transplantation, and, in agreement with this, cyclin D2–transduced myeloid progenitors showed an enhanced proliferative response to cytokines in vitro. Furthermore, cyclin D2–overexpressing myeloid progenitors and their progeny were sustained for longer periods in culture, resulting in enhanced and prolonged granulocyte production in vitro. Thus, overexpression of cyclin D2 confers myeloid progenitors with an enhanced proliferative and granulocyte potential, facilitating rapid myeloid engraftment and rescue of lethally ablated recipients.

Clinical stem-cell sources contain CD8+CD3+ T-cell receptor–negative cells that facilitate bone marrow repopulation with hematopoietic stem cells

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1735-1738 ◽  
Author(s):  
Stephanie Bridenbaugh ◽  
Linda Kenins ◽  
Emilie Bouliong-Pillai ◽  
Christian P. Kalberer ◽  
Elena Shklovskaya ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Hematopoietic Stem ◽  
Cd8 Cells ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Clinical observations in patients undergoing bone marrow transplantation implicate the involvement of CD8+ cells in promoting the stem-cell engraftment process. These findings are supported by mouse transplant studies, which attributed the engraftment-facilitating function to subpopulations of murine CD8+ cells, but the analogous cells in humans have not been identified. Here, we report that clinical stem-cell grafts contain a population of CD8α+CD3ϵ+ T-cell receptor– negative cells with an engraftment facilitating function, named candidate facilitating cells (cFCs). Purified cFC augmented human hematopoiesis in NOD/SCID mice receiving suboptimal doses of human CD34+ cells. In vitro, cFCs cocultured with CD34+ cells increased hematopoietic colony formation, suggesting a direct effect on clonogenic precursors. These results provide evidence for the existence of rare human CD8+CD3+TCR− cells with engraftment facilitating properties, the adoptive transfer of which could improve the therapeutic outcome of stem-cell transplantation.

scholarly journals c-Myc inhibition is critical for HSC expansion and Rad51 expression

2018 ◽  
Author(s):  
Merve Aksoz ◽  
Esra Albayrak ◽  
Galip Servet Aslan ◽  
Raife Dilek Turan ◽  
Lamia Yazgi Alyazici ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Nos Inhibitor ◽  
Kinetics Of

c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence. Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion. Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclin-dependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose derived mesenchymal stem cells, however; it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression. These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.

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