Histone Deacetylase Inhibitors Promote the Ex Vivo Expansion of Cord Blood CD34+ Cells in Serum Free Cultures Accompanied by the Upregulation of Pluripotency Genes

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 345-345 ◽  
Author(s):  
Pratima Chaurasia ◽  
David Gajzer ◽  
Michael Feldman ◽  
Ronald Hoffman
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Ex Vivo Expansion ◽  
Class I ◽  
Aldh Activity ◽  
Serum Free ◽  
Functional Potential ◽  
Cd34 Cells ◽  
Pluripotency Genes

Abstract Abstract 345 Previously, we have reported that various chromatin modifying agents(CMA) can epigenetically alter HSC fate decisions resulting in increased numbers of cord blood(CB)- SCID repopulating cells(SRC) and/or erythroid progenitor cells depending upon the CMA utilized (Blood. 2011,117(17):4632–41). We have hypothesized that treatment of CB CD34+ cells with histone deacetyalse inhibitors(HDACIs) might expand the numbers of CB-SRC and that such cell products could be used as allogeneic stem cell grafts. We developed serum free(SF) and containing(SC) culture conditions and assessed the ability of several HDACIs including valproic acid(VPA), scriptaid(SCR) and CAY10433(C433) to expand CB-SRC Each of these HDACIs were shown to inhibit both class I and II HDACs. The addition of VPA to SF cultures led to a >6,000 fold expansion of CD34+ cells and >25,000-fold expansion of CD34+CD90+ cells as compared to primary cells(PC). By contrast, SC cultures resulted in only a 54-fold expansion of CD34+ cells and a 75-fold expansion of CD34+90+ cells as compared to PC in the presence of VPA.In order to examine the functional characteristics of HDACIs treated CB-CD34+ cells in SF cultures we evaluated aldehyde dehydrogenase activity(ALDH) in these expanded CD34+ cells. A far greater proportion of CD34+ cells were ALDH+ following treatment with VPA in SF cultures as compared to SC cultures (82.2±8.6%, versus 37.0±0.9%). By contrast incubation in the SC cultures induced the generation of greater proportion of ALDH-CD34+ cells as compared to SF cultures (27.0±5.0% versus 3.7±2.0%). These findings indicate that the inclusion of serum to such cultures favors HSC differentiation and eventual HSC depletion while SF conditions favor expansion of more primitive HPC. Purified ALDH+CD34+ cells from SF cultures supplemented with HDACIs contained far greater absolute numbers of BFU-E and CFU-Mix (8.4×107±6.7×107/CB collection) as compared to SC cultures (1.4×107±8.8×106/CB collection; One way ANOVA p=.02). These data indicate that based upon their degree of ALDH activity and functional potential different phenotypically and functionally defined subsets of CD34+ cells are generated in SF as compared to SC cultures. In order to further examine the effects of VPA on the pluripotency of expanded CD34+ cells we examined the expression of Sox2, Oct4 and Nanog in CD34+ cells re-isolated following 7 days of treatment with HDACIs in SF and SC cultures. RNA and protein expression levels of Sox2, Oct4 and Nanog genes were greater in cells generated in SF cultures supplemented with HDACIs than that observed in CD34+ cells cultured in SC. Sox2 and Oct4 bind to the promoter of Nanog and up-regulate Nanog expression. In the SF but not SC cultures VPA treatment resulted in the up-regulation of Nanog indicating the functionality of the Sox2/Oct4 interactions. We also documented using confocal microscopy the elevated expression of the proteins derived from these pluripotency genes both in the cytoplasm and nucleus of CD34+ cells re-isolated after a week of treatment with each of the HDACIs. The functional potential of the re-isolated CD34+ cells following a week of treatment with VPA in SF medium was evaluated by transplanting the expanded cells into NOD/SCID/gamma(c)null mice. SRC capable of multi-lineage hematopoietic differentiation were detected in the marrow of recipients of VPA treated grafts 13-weeks after transplantation (%CD45, 33.5±13.4% with VPA treated grafts versus 13.0±7.7% with grafts generated in the presence of CA). Bone marrow cells from primary recipients receiving HDACI treated CD34+ cells grafts were also capable of generating greater numbers of cells belonging to myeloid and lymphoid lineages(%CD45, 3.3±1.0%) as compared to grafts receiving CA(%CD45, 0.65±0.16%) 15 weeks after their transplantation into secondary recipients. These data indicate that inhibitors of class I and II HDACs under SF culture conditions provide an effective pharmacological approach which can be utilized to promote the ex vivo expansion of functional CB-SRC which are characterized by high ALDH activity and expression of a variety of pluripotency genes. These findings indicate the importance of epigenetic events in determining phenotype and function of dividing SRC in vitro. Disclosures: Hoffman: NYSTEM: Research Funding.

Proliferation and Survival of Mammary Carcinoma Cells Are Influenced by Culture Conditions Used for Ex Vivo Expansion of CD34+Blood Progenitor Cells

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 746-755 ◽  
Author(s):  
A. Spyridonidis ◽  
W. Bernhardt ◽  
D. Behringer ◽  
G. Köhler ◽  
M. Azemar ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cells ◽  
Mammary Carcinoma ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Ex Vivo Expansion ◽  
Carcinoma Cells ◽  
Serum Free ◽  
Cellular Contact ◽  
Mammary Carcinoma Cells

Abstract Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34+ blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1β (IL-1β), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34+ BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-β1 (TGF-β1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1β + IL-3 + IL-6 + EPO, but was suppressed by TGF-β1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 × 105/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34+ BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34+ BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.

scholarly journals Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression

2001 ◽  
Vol 16 (1) ◽  
pp. 20 ◽  
Author(s):  
Ju Young Seoh ◽  
Hae Young Park ◽  
Wha Soon Chung ◽  
Seung Cheol Kim ◽  
Myong Joon Hahn ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Ex Vivo Expansion ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Late Antigen ◽  
Cord Blood Cd34 ◽  
Cell Cycling

Biomimetic Macroporous PCL Scaffolds for Ex Vivo Expansion of Cord Blood-Derived CD34+Cells with Feeder Cells Support

2017 ◽  
Vol 17 (8) ◽  
pp. 1700054 ◽  
Author(s):  
Xiuwei Pan ◽  
Qiong Sun ◽  
Yuanhao Zhang ◽  
Haibo Cai ◽  
Yun Gao ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Feeder Cells ◽  
Cd34 Cells

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.

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.

Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4954-4954
Author(s):  
Ronald L. Brown ◽  
J. Zhang ◽  
L. Qiu ◽  
A. Nett ◽  
G. Almeida-Porada ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Cell Populations ◽  
Multilineage Differentiation ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Cytokine Combination ◽  
Cells Cultured

Abstract Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.

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.

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