Expansion Of Cord Blood Cells Research Articles

Generation of Megakaryocytes and Platelets from Valproic Acid Expanded Human Cord Blood CD34+ Cells in Culture

Currently in the US alone there are more than 2 million units of platelets transfused each year. The transfusion of platelets can be crucial life saving measures for patients undergoing treatment for leukemia and other cancers. However, volunteer donors are the only source of platelets for transfusions. The objectives of our studies are to further refine our multi-step culture strategies to expand human umbilical cord blood (CB) derived megakaryocyte (MK) progenitors, and augment functional MK/platelet yields. We used serum-free culture to expand stem/progenitor cells over 5 to 7 days from CB and growth factor mobilized peripheral blood (mPB) CD34+ cells using valproic acid (VPA), a histone deacetylase inhibitor (HDAC). VPA-expanded CB cells were assayed for MK progenitors by flow cytometry and CFU-MK assays. Compared to mPB, CB CD34+ cells yielded relatively more CD34+CD90+ cells (>1.6 fold), yet the absolute numbers of CD34+CD41a+ MK progenitors were comparable (CB 7.18 ± 1.19 x 105/well vs. mPB 7.55 ± 1.14 x 105/well). The CFU-MK colony yield from mPB was relatively lower than CB (CB 32.31 ± 9.57 x 103/well vs. mPB 11.3 ± 5.74 x 103/well, not significant). Extending the CB cultures up to 7 days resulted in notably greater fold increase of CD34+CD90+ primitive stem/progenitor cells from prior to expansion (D5 VPA 75.77 ± 14.83 vs. D7 VPA 146.26 ± 28.52-fold increase of CD34+CD90+ cells; p = 0.07, n = 4) as well as increase in CD34+CD41a+ MKs (D5 VPA 1.02 ± 0.24 X 105/well, n = 8, vs. D7 VPA 2.29 ± 0.85 X 105/well, n= 6, p = 0.04) and slightly higher number of CFU-MK colonies. We then estimated CD41a+ MK cell yield per initial single CD34+ CB cell plated to initiate expansion culture. VPA-expanded CD34+ cells were further cultured in the presence of various small molecules including stem reginin 1 (SR1), UM171, VPA, and nicotinamide. The yield of CD41a+ MKs from a single CD34+ cell plated increased with time. Having UM171 or VPA in extended cultures is favorable to generate high yield CD41+ MKs. Interestingly, a single CD34+ cell following VPA-expansion yielded up to 2,060.32 ± 631.48 (mean ± SE, n= 6) CD41a+ MKs after 17 days of culture. If we plated every single cell from a CB unit containing 5 million CD34+ cells in VPA expansion culture and continued the cultures for 17 days, 10.3 billion CD41a+ cells (10.3 x 109) could be generated. If we are capable of producing about 50 platelets per MK plated using optimized culture conditions, this would yield almost 2 units of transfusible platelets (5.15 x 1011) for patients. Using cultures of CB CD34+ cells, the relative expression levels of 12 genes were assessed by real-time qRT-PCR. Genes tested were classified according to the relative abundance of transcripts for the erythroid (EPOR and KLF1 ) and MK (AML1 , FLI1 , GABPA , and MPL ) lineages, as well as genes common to both lineages (GATA1 , NFE2 , TAL1 , GATA2 , MYB , and LMO2 ). Interestingly in MK inductive culture, VPA treated D14 samples displayed relatively lower transcript levels of erythroid genes including EPOR and KLF1 and higher expression of MK specific gene Fli1. After further differentiation at day 20 the expression of MK specific gene FLI1 achieved the highest level of expression (D0 to D7 1.84 ± 0.3; D0 to D20 3.52 ± 0.79-fold increase) and erythroid gene KLF1 had the lowest level of expression (D0 to D7 12.15 ± 1.72; D0 to D20 5.76 ± 3.77-fold increase) relative to pre-culture samples. Taken together, MK specific gene transcript levels increased while erythroid specific gene expression decreased following extended culture of CD34+ cells. These results suggest that VPA expanded CB cells maintain MK lineage commitment during extended culture. The MKs generated in culture appear to be relatively immature and only 21.44 ± 2.99 % (n = 4) of CD41a+ MKs co-expressed CD42b. However, in optimal MK condition the VPA expanded CB cells were capable of generating mature MKs (CD41a+/CD42b+) leading up to 3 to 7 times more CD41a+CD42b+ MKs in comparison to unexpanded CB CD34+ cells. Platelets generated per CD41a+CD42b+ MKs from VPA-expanded CD34+ cells were also 2 to 4 times greater than unexpanded CD34+ cells. Our current MK generation strategies from VPA-expanded CB cells, once further scaled up and optimized for MK maturation/platelet generation, has the potential to generate about two units of platelets for transfusion. DisclosuresNo relevant conflicts of interest to declare.

Comparison of Outcomes between HSCT Donor Sources for Pediatric Patients with Hematologic Malignancies

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for pediatric patients with hematologic malignancies. Historically, matched sibling donors (MSD) have been the preferred donor source given ease of availability and lower rates of graft-versus-host-disease (GVHD). However, only 30% of patients have a MSD and relapse rates are high after MSD HSCT, raising the question of best donor choice. As conditioning regimens evolve, GVHD management improves and supportive care advances, it is important to evaluate the role of donor source on short and long-term clinical outcomes to inform donor selection. We performed a single-center retrospective analysis comparing post-HSCT outcomes in a cohort of pediatric patients undergoing MSD, matched unrelated donor (MUD), and umbilical cord blood (CB) transplants from 2006 to 2018. Methods: A retrospective analysis was performed on an IRB-approved protocol through Fred Hutchinson Cancer Research Center. 232 patients were included who received MSD (n=56), MUD (n=89) or CB (n=87) transplants. Of note, 24 CB patients received expanded CB cells in addition to unmanipulated unit(s). GVHD prophylaxis in all patients consisted of a calcineurin inhibitor and MMF or methotrexate. The vast majority received a high-intensity conditioning regimen (86%, 96%, and 82% respectively for MSD, MUD and CB). Overall survival (OS) and disease-free survival (DFS) were evaluated using the Kaplan-Meier method. Probabilities of non-relapse mortality (NRM), relapse, and acute GVHD were evaluated using cumulative incidence (CI) estimates with appropriate competing risks. The Cox regression model was used for adjusted analysis for age, year of transplant, sex, CMV status, MRD status, disease risk, and conditioning regimen. Results: Patient/treatment/donor demographics are shown in Table 1. Median follow-up was 2.6, 3.7 and 3.1 years for MSD, MUD and CB respectively. Patient diagnosis, disease risk, gender, age, and CMV serology were balanced between groups. CI of engraftment was similar as well, with only one graft failure in the MUD group (Fig 1). Median time to platelet recovery was significantly faster in MUD and MSD groups as compared to the CB group (p<0.0001; p<0.0001). There was no difference in unadjusted 5 year OS (Fig 2) and NRM (Fig 3) between groups. Five-year DFS was significantly higher in CB v. MSD (71% v. 54%, p=0.03) but not between CB v. MUD (71% v. 61%, p=0.18) or between MSD and MUD (p=0.38) (Fig 4). The CI of relapse at 5 years was 20% for CB patients, significantly lower than that of MSD recipients (49%, p=0.003) and not different from MUD (32%, p=0.11) (Fig 5). After adjusted analysis, the risk of DFS failures was higher among recipients of MSD than CB [HR: 1.88 (CI 95%: 1.01-3.47), p=0.04]. When CB versus MUD groups were compared, there was a trend of higher risk of DFS failures in the MUD group [HR: 1.28 (CI 95%: 0.96-1.66), p=0.09]. No difference was observed in risk of DFS failures between MSD versus MUD groups [HR:0.77 (CI 95%: 0.44-1.4), p=0.43]. Incidence of grade II-IV acute GVHD was 90% (95%CI: 82-95%), 75% (95%CI: 61-84%) and 88% (95%CI: 80-94%), for CB, MSD and MUD, respectively (p=0.25). Incidence of grade III-IV acute GVHD was 33% (95%CI: 23-43%) for CB, 9% (95%CI: 3-18%) for MSD, and 11% (95%CI: 5-18%) for MUD. Incidence of grade III-IV was significantly higher for the CB group compared to the other groups (p=0.001); however, nearly 60% of CB recipients with grade III-IV acute GVHD were diagnosed before engraftment had occurred and in retrospect met criteria for pre-engraftment syndrome. Among surviving patients, 23 CB recipients developed chronic GVHD (26% - 15 mild, 4 moderate, 4 severe) as compared to 14 MSD patients (25% - 13 mild, 1 moderate) and 40 MUD patients (45% - 27 mild, 11 moderate, 2 severe). Conclusions: Our data demonstrate no difference in unadjusted OS between MSD, MUD and CB recipients. Importantly, despite this equivalence, 5-year DFS was significantly better in the CB v. MSD group, reflecting the lower relapse rate observed in CB patients and seen previously by us and others. CB continues to be viewed as an "alternative" donor for HSCT due to the low stem cell dose in a CB graft resulting in delayed neutrophil recovery, primary graft failure and increased NRM. However, this was not observed herein, supporting the use of CB for pediatric HSCT perhaps especially in patients at high risk of post-transplant relapse. Disclosures Milano: ExCellThera: Research Funding; Amgen: Research Funding. Delaney:Nohla Therapeutics: Employment, Equity Ownership; Biolife Solutions: Membership on an entity's Board of Directors or advisory committees. Bleakley:HighPass Biotherapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Conditioned Medium Represents a Useful Solution to Increase the Expansion of Multipotent Progenitors with Strong Platelet Engraftment Activity

Delayed neutrophil and platelet engraftment is a significant issue of cord blood (CB) transplantation. Ex vivo expansion of CB hematopoietic stem and progenitor cells (HSPC) before infusion has been shown to accelerate hematopoietic recovery in patients. Recently, we reported that serum free medium (SFM) conditioned with osteoblasts derived from human bone marrow (BM) mesenchymal stromal cells, referred as M-OST CM, was superior to SFM or MSC CM for the expansion of CB CD34+ cells, and that HSPC expanded in M-OST CM provided better platelet engraftment. Since large number of expanded cells were transplanted in the original study, it was not possible to estimate the increased expansion of HSPC with short-term (ST) and long-term (LT) thrombopoietic and BM engraftment activities. The objectives herein were to investigate these shortcomings using limit dilution analysis (LDA) in transplantation assay and to investigate the cellular mechanisms at play.M-OST CM was prepared by conditioning SFM with immature M-OST overnight. CB CD34+ cells were expanded in M-OST CM or in SFM (defined as control) for 7 days with SCF, FL and TPO. CB cell expansion was significantly greater in M-OST CM cultures vs. SFM control (2.4 ±0.9 fold, mean ± SD, n=4, p=0.01). LDA transplantation assays were done by infusing the progeny of 500-8000 CD34+ cells in NSG mice. First, we compared the ST (< 31 days) and LT (˃ 100 days) thrombopoietic activities of expanded HSPC by measuring circulating human platelets (hPLT). The threshold for hPLT engraftment was set above the mean background level measured in control mice + 1SD. The median ST levels of hPLT in M-OST mice tended to be greater (2.5-fold, p˃0.05) in M-OST recipients (21 mice/condition, n=2). The frequency of ST hPLT HSPC estimated by LDA was 3.4 ±0.2 fold higher in M-OST CM cultures though the difference vs. control was not significant (p=0.11). LT hPLT levels were significantly greater in M-OST recipients (median 33 vs. 8 hPLT/uL blood, p=0.0027). Consistent with this, the frequency of HSPC with LT hPLT engraftment was increased in M-OST CM cultures (3.5±0.8 fold, p<0.04). Considering the differences in cell expansion, the net expansion of HSPC with ST and LT hPLT engraftment were raised by 5.5 ±1.7 and 6.0 ±3.4 fold in M-OST CM cultures vs. control (n=2). Next, LT human BM engraftment was analyzed at week 16. Preliminary results (13 mice/condition) suggest that the frequency of LT Scid repopulating cells (SRC) was increased by 27% in the M-OST CM culture vs. SFM control (frequency of 1/2878 vs. 1/3626 of day 0 starting cell).Next, we set to determine how M-OST CM increases the thrombopoietic activity of expanded CB HSPC. First, cytometry analysis (CD34, CD38, CD45RA, CD90 and CD123) revealed that M-OST CM preferentially increased the expansion of common myeloid progenitors (CMP, 8-fold, p=0.2, n=3), megakaryocyte-erythroid progenitors (MEP, 7-fold, p=0.02) and granulocyte-macrophage progenitors (GMP, 9-fold, p=0.02) vs. SFM control. Expansion of HSC-enriched cells was unchanged while that of multipotent progenitors (MPP) was reduced 2-fold (p<0.05). We set to confirm these results by culturing purified primary CB HSPC subsets in M-OST CM or SFM; M-OST CM induced greater expansions of MEP (3-fold), GMP (˃10-fold) while expansion in MPP cultures was greater with SFM control (1.5-fold). No growth was noted with the HSC and CMP cultures likely due to low sort yields.To complement these findings, we measured the expansion of myeloid CFU progenitors and long term culture-initiating cells (LTC-IC) by LDA. The total number of CFU was increased 2.4-fold (<0.02, n=4) by M-OST CM due mostly to increased expansion of CFU-G/GM colonies (2-fold, p<0.05) and BFU-E (2-fold, p=0.05). M-OST CM also sustained a 3.4-fold increase in LTC-IC expansion vs. SFM culture, though this finding remains to be confirmed in ongoing experiments.Finally, we investigated the effect of M-OST CM on the chemotaxis of HSPC toward SDF-1 since we previously reported increased expression of its receptor CXCR4 on CB cells in M-OST CM cultures. M-OST CM HSPC showed a modest 15% increase in migration vs. SFM control (n=4, p=0.10).In conclusion, our results demonstrate that the ST and LT hPLT engraftment activities of ex vivo expanded CB HSPC can be increased 5-6 folds by the use of M-OST CM due to the expansion of immature CB HSPC subsets including perhaps LT SRC. Whether M-OST CM can also modulate the homing activity of HSPC remains unclear. DisclosuresNo relevant conflicts of interest to declare.

No evidence of clonal dominance after transplant of HOXB4-expanded cord blood cells in a nonhuman primate model

Umbilical cord blood transplant continues to increase in prevalence as a treatment option for various hematopoietic and immune disorders. Because of the limited number of cells available in a single cord blood unit, investigators have explored methods of increasing cell dose before transplant, including overexpression of the homeobox B4 (HOXB4) transcription factor. We have previously reported the development of leukemia in several nonhuman primate (NHP) subjects transplanted with HOXB4-expanded bone marrow cells at approximately 2 years posttransplant. Here, we provide long-term data for a NHP receiving a HOXB4-expanded cord blood graft. Longitudinal follow-up included gene marking analysis, complete blood counts, morphologic/pathologic assessment, phenotypic analysis of subsets, and retroviral integration site analysis. In each of these independent assays, we saw no indication of clonal dominance, and all signs pointed toward normal, healthy hematopoiesis. Furthermore, in-depth clonal analysis of an animal that developed leukemia after transplantation of HOXB4-modified bone marrow cells showed that dominant clones could be detected as early as 6 months posttransplant using the genomic analysis technique detailed here. Parallel analysis of the cord blood transplant macaque showed no such sites. These findings demonstrate the ability to study the use of gene-modified and expanded cord blood cells in a NHP model.

Real‐time monitoring and control of soluble signaling factors enables enhanced progenitor cell outputs from human cord blood stem cell cultures

Monitoring and control of primary cell cultures is challenging as they are heterogenous and dynamically complex systems. Feedback signaling proteins produced from off-target cell populations can accumulate, inhibiting the production of the desired cell populations. Although culture strategies have been developed to reduce feedback inhibition, they are typically optimized for a narrow range of process parameters and do not allow for a dynamically regulated response. Here we describe the development of a microbead-based process control system for the monitoring and control of endogenously produced signaling factors. This system uses quantum dot barcoded microbeads to assay endogenously produced signaling proteins in the culture media, allowing for the dynamic manipulation of protein concentrations. This monitoring system was incorporated into a fed-batch bioreactor to regulate the accumulation of TGF-β1 in an umbilical cord blood cell expansion system. By maintaining the concentration of TGF-β1 below an upper threshold throughout the culture, we demonstrate enhanced ex vivo expansion of hematopoietic progenitor cells at higher input cell densities and over longer culture periods. This study demonstrates the potential of a fully automated and integrated real-time control strategy in stem cell culture systems, and provides a powerful strategy to achieve highly regulated and intensified in vitro cell manufacturing systems. Biotechnol. Bioeng. 2014;111: 1258–1264. © 2013 The Authors Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

CD34+ Expansion With Delta-1 and HOXB4 Promotes Rapid Engraftment and Transfusion Independence in a Macaca nemestrina Cord Blood Transplant Model

Umbilical cord blood (CB) transplantation is a promising therapeutic approach but continues to be associated with delayed engraftment and infections. Here, we explored in our macaque CB transplant model expansion and engraftment kinetics of cells expanded with the combination of HOXB4 and Delta-1. CB cells were divided into two equal fractions; one fraction was transduced with HOXB4 yellow fluorescent protein (YFP) and expanded on control OP9 cells, and the other was transduced with HOXB4 green fluorescent protein (GFP) and expanded on Delta-expressing OP9 cells (OP9-DL1). Both fractions were transplanted into myeloablated subjects. Neutrophil and platelet recovery occurred within 7 and 19 days respectively, which was significantly earlier than in our previous study using cells expanded with HOXB4 alone, which resulted in neutrophil recovery within 12 days (P = 0.05) and platelet recovery within 37 days (P = 0.02). Furthermore, two of three animals in the current study remained fully transfusion-independent after transplantation. By day 30, reconstitution of lymphocytes was significantly greater with the HOXB4/OP9-DL1 expanded cells in all animals (P = 0.05). In conclusion, our data show that the combination of OP9-DL1 and HOXB4 can result in increased numbers of repopulating cells, thus leading to rapid engraftment and transfusion independence in macaques transplanted with autologous, expanded CB cells.

Exploring the use of expanded erythroid cells for autologous transfusion for anemia of prematurity

Autologous cord blood (CB) red blood cells (RBCs) can partly substitute transfusion needs in premature infants suffering from anemia. To explore whether expanded CB cells could provide additional autologous cells suitable for transfusion, we set up a simple one-step protocol to expand premature CB cells. CB buffy coat cells and isolated CD34-positive (CD34(pos) ) cells from premature and full-term CB and adult blood were tested with several combinations of growth factors while omitting xenogeneic proteins from the culture medium. Cell differentiation was analyzed serially during 21 days using flow cytometry, progenitor assays, and high-performance liquid chromatography. Expanded CB buffy coat cells resulted in a threefold higher number of erythroblasts than the isolated CD34(pos) cells. However, the RBCs contaminating the buffy coat remained present during the culture with uncertain quality. Premature and full-term CB CD34(pos) cells had similar fold expansion capacity and erythroid differentiation. With the use of interleukin-3, stem cell factor, and erythropoietin, the fold increases of all CD34(pos) cell sources were similar: CB 3942 ± 1554, adult peripheral mobilized blood 4702 ± 1826, and bone marrow (BM) 4143 ± 1908. The proportion of CD235a expression indicating erythroblast presence on Day 21 was slightly higher in the adult CD34(pos) cell sources: peripheral blood stem cells (96.7 ± 0.8%) and BM (98.9 ± 0.5%) compared to CB (87.7 ± 2.7%; p = 0.002). We were not able to induce further erythroid maturation in vitro. This explorative study showed that fairly pure autologous erythroid-expanded cell populations could be obtained by a simple culture method, which should be optimized. Future challenges comprise obtaining ex vivo enucleation of RBCs with the use of a minimal manipulating approach, which can add up to autologous RBCs derived from CB in the treatment of anemia of prematurity.

Cryopreservation of hematopoietic stem and progenitor cells amplified ex vivo from cord blood CD34+ cells

Our ex vivo expansion procedure starting from cord blood (CB) CD34+ cells enabled expansion of committed progenitors (CPs) without a negative impact on hematopoietic stem cells (HSCs) exhibiting both short- and long-term repopulating capacity. Upgraded to clinical scale (Macopharma HP01 in the presence of stem cell factor, FLT3-L [100 ng/mL each], granulocyte-colony-stimulating factor [10 ng/mL], and thrombopoietin [20 ng/mL]), it is being used for an ongoing clinical trial (adult allogeneic context) yielding promising preliminary results. Transplantation of ex vivo expanded CB cells is becoming a reality, while the issue of expanded cells' cryopreservation emerges as an option that allows the conservation of the product for transportation and future use. Here, we investigated whether it is possible to maintain the functional HSC and CP properties after freezing and thawing of expanded cells. We compared cryopreservation efficiency of the ex vivo expanded CB cells using the standard protocol (freezing solution human serum albumin (HSA)-dimethyl sulfoxide [DMSO]) with the newly designed protocol based on an enriched freezing solution (HP01-DMSO) with respect to the viability index, number of CD34+ and total cells, and recovery of CPs (colony-forming units) and HSCs (NOG/Scid/gamma-null mice engraftment). Cryopreservation and thawing of expanded CB cells using the "standard" procedure (HSA-DMSO) reduced recovery of the CPs (40%) and HSCs (drastically decreasing engraftment capacity). HP01-based protocol resulted in improvement of preservation of both CPs (>60%) and HSCs (nonaltered engraftment capacities). Functional maintenance of the expanded graft by cryopreservation is feasible in conditions compatible with human cell therapy requirements.

Cotransplantation of Ex Vivo Expanded and Unexpanded Cord Blood Units in Immunodeficient Mice Using Insulin Growth Factor Binding Protein-2–Augmented Mesenchymal Cell Cocultures

Ex vivo expansion of cord blood (CB) hematopoietic stem cells and cotransplantation of 2 CB units (CBUs) could enhance the applicability of CB transplantation in adult patients. We report an immunodeficient mouse model for cotransplantation of ex vivo expanded and unexpanded human CB, showing enhanced CB engraftment and provide proof of concept for this transplantation strategy as a means of overcoming the limiting cell numbers in each CBU. CBUs were expanded in serum-free medium supplemented with stem cell factor, Flt-3 ligand, thrombopoietin, and insulin growth factor binding protein-2 together with mesenchymal stromal cell coculture. Unexpanded and expanded CB cells were cotransplanted by tail vein injection into 45 sublethally irradiated nonobese diabetic SCID-IL2γ(-/-) (NSG) mice. Submandibular bleeding was performed monthly, and mice were sacrificed 4 months after transplantation to analyze for human hematopoietic engraftment. Expansion of non-CD34(+) selected CB cells yielded 40-fold expansion of CD34(+) cells and 3.1-fold expansion of hematopoietic stem cells based on limiting dilution analysis of NSG engraftment. Mice receiving expanded grafts exhibited 4.30% human cell repopulation, compared with 0.92% in mice receiving only unexpanded grafts at equivalent starting cell doses, even though the unexpanded graft predominated in long-term hematopoiesis (P = .07). Ex vivo expanded grafts with lower initiating cell doses also showed equivalent engraftment to unexpanded grafts with higher cell dose (8.0% versus 7.9%; P = .93). In conclusion, ex vivo expansion resulted in enhanced CB engraftment despite eventual rejection by the unexpanded CBU.

Possible Reduced Immunogenicity of Chromatin Modifying Agents Expanded Cord Blood Grafts Is Likely Due to STAT3 Inhibition,

Abstract 4016The suboptimal number of hematopoietic stem cells (HSC) and relatively high incidence of graft failure following umbilical cord blood (CB) transplantation in adult patients remain as major obstacles to the widespread use of CB grafts. Using chromatin modifying agents (CMAs), including 5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA), we have developed a culture strategy that permits 10 to 12 fold expansion of CB CD34+CD90+ cells containing in vivo repopulating cells (Araki et al. Blood 2007, Exp Hematol 2009). It is important that potential ex vivo expansion strategies not increase the capacity of the graft to mount an immune response to an allogeneic host. We have therefore assessed the allo-stimulatory capacity of expanded grafts following 9 days of culture examining immunophenotypic markers of dendritic cell (DC) commitment and also co-stimulatory molecules expressed on CMA-expanded CB cells. 5azaD/TSA-expanded cells co-expressing antigen presenting cell (APC) markers such as HLA-DR/CD86 comprise 17 ± 7% of the graft in contrast to 38 ± 9% in CB cells expanded in control culture and 2 ± 1% of primary CD34+ cells (n=3). Expression of HLA-DR/CD11c was only 3% in 5azaD/TSA-expanded cells in contrast to 37% in CB controls and 60% in primary CD34+ cells. Expression of the co-stimulatory molecule CD40 was not altered. We next performed mixed lymphocyte cultures (MLC), co-culturing irradiated primary or expanded CB cells with allogeneic peripheral blood T cells. The immune stimulatory capacity was measured by thymidine uptake, with CPM values of 3,213 ± 2205, 26,461 ± 670, and 2,859 ± 761 in primary CB cells, CB cells grown in control culture and 5azaD/TSA-expanded cells respectively (control vs. 5azaD/TSA p=0.001, 5azaD/TSA vs D0 p=0.893, n=3). The MLC results suggest that 5azaD/TSA-expanded CB grafts possess 9-fold less allo-stimulatory capacity than control cultures but are similar to primary CB cells. We hypothesized that this reduced allo-stimulatory capacity of 5azaD/TSA-expanded grafts could be due to three possible reasons:1) CMA-induced inhibition of expression of co-stimulatory molecules; 2) inhibition of the differentiation of CD34+ cells to APC; and/or 3) apoptosis of APC due to exposure to CMA. It has been previously shown that activation of the transcription factor STAT3 is indispensable for differentiation of DC from HSC. To study the molecular mechanisms involved in the reduced allo-stimulatory capacity of CMA expanded grafts, transcript levels of several genes, including STAT3 were determined by real time quantitative RT PCR. Interestingly the transcript levels of STAT3 were 2.5-fold higher in 5azaD/TSA-expanded CD34+ cells compared to control cultures. Furthermore the expression of two known inhibitors of STAT3, P21 and GATA1, were upregulated 10.5- and 6.8-fold respectively in 5azaD/TSA-expanded CD34+ cells. Therefore although STAT3 gene expression is increased in 5azaD/TSA-expanded cells it is likely inhibited by the increased levels of the inhibitors P21 and GATA1. In support of this we observed a reduced capacity for DC generation in cultures from 5azaD/TSA-expanded cells compared to control cultures. As expected, a LINE-1 assay, a surrogate marker of global methylation, shows that CpG methylation in 5azaD/TSA-expanded CD34+ cells is reduced to 66% compared to 78% in control cultures. Notably however the decrease in methylation specifically affects CpGs near the promoter site of GATA1 (control 82%, 5azaD/TSA 66%), but not STAT3 or P21. Furthermore, SHP1, a tyrosine phosphatase known to inhibit STAT3 activation, is also upregulated 2-fold in 5azaD/TSA-expanded CD34+ cells compared to control cultures. SHP1 transcription is regulated both by promoter methylation and by interactions with c-MYC, however no appreciable difference in c-MYC expression was detectable between 5azaD/TSA-expanded and control cultures suggesting that SHP1 upregulation in 5azaD/TSA-expanded CD34+ cells also involves promoter hypomethylation. Taken together, our studies indicate that the allo-stimulatory capacity of CB cells expanded in 5azaD/TSA- is reduced rather than increased, likely due to inhibition of an early stage of DC differentiation from CD34+ cells, which is mediated by inhibition of STAT3 activation. Reduced allo-stimulatory capacity of CMA expanded CB grafts may provide benefits by potentially allowing transplantation with less stringent HLA matching. Disclosures:No relevant conflicts of interest to declare.

Ex Vivo Expansion of Human Mobilized Peripheral Blood Stem Cells Using Epigenetic Modifiers

Abstract 1920Currently, a significant percentage of hematopoietic stem cell (HSC) transplantations are being performed using growth factor mobilized peripheral blood (MPB) grafts. Unfortunately, about 5 to 40% of patients are unable to benefit from HSC transplantation due to failure to mobilize and harvest an adequate graft (> 2 × 106 CD34+ cells/kg). Epigenetic modifications are thought to be important in determining the fate of HSC including self renewal and differentiation. We have previously demonstrated that sequential addition of chromatin modifying agents (CMA), 5-aza-2’-deoxyctidine (5azaD) and trichostatin A (TSA), is capable of expanding transplantable HSC 7-fold from human cord blood (CB), likely by preventing the silencing of genes which promote HSC self renewal divisions (Araki et al. Blood 2007). Using the same protocol we have also previously shown that 5azaD/TSA can expand CD34+CD90+ cells containing in vivo repopulating capacity from human bone marrow (BM) 2.5-fold (Milhem et al. Blood 2004). The objectives of our current studies were to assess whether CMA can also expand HSCs present in MPB. In order to test this hypothesis, CD34+ cells were isolated from MPB products from three healthy donors and were expanded ex vivo using 5azaD/TSA for 9 days as described previously (Araki et al. Blood 2007). Following culture, expansion of primitive CD34+CD90+ cells, colony forming unit mixed lineages (CFU-mix), and long term (5 weeks) cobblestone area forming cells (CAFC) were assessed. A 3.74 ± 0.77 fold expansion of CD34+CD90+ cells was observed in 5azaD/TSA expanded MPB cells while only a 0.93 ± 0.23 fold expansion was observed in control cultures (p = 0.025). The 5azaD/TSA expanded MPB cells had a 10.1-fold increase in the number of CFU-mix in comparison to no expansion in the control cultures (p = 0.0055). A 2.26-fold expansion of CAFC numbers was observed in 5azaD/TSA expanded MPB cells in comparison to 0.19-fold expansion in control cultures. Taken together, our data indicate that 5azaD/TSA can expand MPB CD34+CD90+ cells 3.74-fold which also possess the functional capacity to generate primitive CFU-mix and long term CAFCs. This expansion of primitive MPB CD34+CD90+ cells appears to be at an intermediate level (3.74 fold) in comparison to BM and CB which had 2.5-fold and 10.5-fold expansion, respectively. We have previously demonstrated that CD34+CD90+ expanded CB cells are exclusively responsible for reconstituting blood cells following transplantation (Araki et al. Exp Hematol 2006). Currently, the frequency of in vivo repopulating units for CMA expanded MPB is being determined in contrast to expanded BM and CB cells. However, it remains to be investigated what determines the limit for ex vivo expansion of HSC by epigenetic modifiers based on their ontogeny. Towards this goal we analyzed transcription levels of several genes implicated for HSC self renewal/expansion including HoxB4, GATA 2, and Ezh2, which were compared between MPB cells prior to and following expansion in 5azaD/TSA or control cultures. Significantly higher transcript levels were detected for HoxB4 (p = 0.003), GATA 2 (p = 0.0002), and Ezh2 (p = 0.0001) by real time quantitative RT PCR in the 5azaD/TSA expanded MPB graft in comparison to control cultures. Interestingly the transcript levels of HoxB4 and GATA 2 but not Ezh2 were significantly lower in expanded cells in contrast to unmanipulated primary MPB cells. This is in sharp contrast to our earlier results from CB in which 5azaD/TSA expanded cells displayed much higher transcript levels of HoxB4 and GATA 2 compared to primary unmanipulated CB cells. Previously we have demonstrated that environmental conditions can influence the degree of expansion of transplantable HSC from CB (Araki et al. Exp Hematol 2009). Using the same protocol we expanded MPB cells in the presence or absence of CMA using either optimal (SCF, TPO, FLT3L) or suboptimal cytokine cocktails (SCF, TPO, FLT3L with IL-3 and IL-6). Interestingly, unlike CB cells no significant difference in expansion between the two cytokine groups with or without CMA was observed (4.5 versus 4.3-fold expansion of CD34+CD90+ cells, respectively). Corresponding to this, transcript levels of HoxB4 and Ezh2 did not vary between MPB cells expanded with 5azaD/TSA in the two different cytokine environments. Our studies have the potential to be used to expand HSC from poor mobilizers in order to optimize MPB grafts for transplantation. Disclosures:No relevant conflicts of interest to declare.

Milestones in umbilical cord blood transplantation

Much has been learned about umbilical cord blood (UCB) since the first human cord blood transplant was performed back in 1988. Cord blood banks have been established worldwide for the collection, cryopreservation and distribution of UCB for allogeneic haematopoietic stem cell transplantation. UCB has now become one of the most commonly used sources of haematopoietic stem cells for allogeneic transplantation. Today, a global network of cord blood banks and transplant centres has been established with a large common inventory, allowing for more than 20000 transplants worldwide in children and adults with severe haematological diseases. Several studies have been published on UCB transplant, assessing risk factors such as cell dose and human leucocyte antigen mismatch. New strategies are ongoing to facilitate engraftment and reduce transplant-related mortality and include the use of reduced-intensity conditioning regimen, intra-bone injection of cord blood cells, double cord blood transplants or ex vivo expansion of cord blood cells. The absence of ethical concern and the unlimited supply of cells explain the increasing interest of using UCB for developing regenerative medicine.

Combination of HOXB4 and Delta-1 ligand improves expansion of cord blood cells

AbstractUmbilical cord blood (UCB) is an attractive cell source for hematopoietic cell transplantation (HCT). Here we examine whether the combination of homeobox B4 (HOXB4) and Delta-1 ligand (DL) synergize when used together. Monkey and human UCB CD34+ cells were transduced with a HOXB4-expressing gammaretroviral vector and cultured with DL. Individual and combined effects of HOXB4 and DL were assessed by colony-forming unit assays, flow cytometry, and nonobese diabetic/severe combined immune deficienct mouse transplantation. The presence of DL yielded higher percentage of CD34+ and CD7+ cells and lower percentages of CD14+ cells than non-DL cultures. Furthermore, HOXB4 yielded higher percentages of CD34+ and CD14+ cells than non-HOXB4 cultures. Interestingly, coculture with DL-expressing OP9 cells resulted in better maintenance of HOXB4 than culture in DL-conditioned medium. Culture of HOXB4-transduced human cells in the presence of DL yielded enhanced generation of repopulating cells with higher levels of engraftment of human CD45+, CD34+, CD3+, CD20+, and CD41+ cells compared with either factor individually. Our results demonstrate enhanced generation of hematopoietic progenitors by combining HOXB4 and DL; addition of DL further enhances expansion of multipotent cells capable of repopulating lymphoid and megakaryocyte lineages, which is not observed with HOXB4 alone.

Characterization of the Impact of Culture on the Thrombopoietic Potential of Cord Blood Progenitors.

AbstractAbstract 3710Co-transplantation of ex vivo expanded progenitors with unmanipulated hematopoietic stem cells improves the recovery of neutrophils while that of platelets remains problematic especially for cord blood (CB) transplantation. The lack of efficacy on platelet recovery could be the result of poor expansion of megakaryocyte progenitors (Mk-P) and Mks induced by the cytokine cocktails currently used, or to the lack of optimization of this procedure. Therefore, we recently developed two cytokine cocktails by statistical design of experiment, OMPC and BS1 for the expansion of CB Mk-P and for the differentiation of CD34+ cells into Mk, respectively. Our objectives were now to compare the short term thrombopoietic potential of CB cells expanded with these cocktails to that of unexpanded cells, and to investigate the impact of two culture parameters, the temperature of incubation and the length of expansion, on platelets recovery.CB CD34+ cells were expanded in serum-free medium with the cocktail BS1 (SCF, TPO, IL-6, IL-9) or OMPC (SCF, FL, TPO). The total progeny of 130,000 expanded CD34+ cells were transplanted into irradiated NOD/SCID mice (9 mice/group). Unexpanded CD34+ cells were transplanted as control to determine whether this procedure procures any advantages to platelet recovery. Human platelets (hPLT) were monitored in the peripheral blood by cytometry, as well as human bone marrow (BM) engraftment.Since we previously showed that Mk expansion and Mk purity can be improved by incubating CB cultures at 39°C, we first assessed the impact of mild hyperthermia (MH) on the thrombopoietic potential of OMPC-expanded cells. hPLTs levels 4 days post-transplant (PT) achieved with cells expanded at 37°C were low and insignificant vs the PBS-control group (p>0.05). In contrast, hPLT levels were 20-fold greater in mice transplanted with cells cultured at 39°C (median of 0.7 vs 13.7 hPLT/uL, respectively p<0.002). No differences were then observed up to 36 days-PT, at which time hPLT levels became greater (8.5-fold) in mice injected with cells expanded at 37°C (p<0.04). Next, the short-term hPLT potentials of CB cells expanded for 6 days were compared to that of unmanipulated CD34+ cells. Only low and insignificant hPLT levels (p>0.05 vs PBS-control group) were detected 4- and 7 days-PT with the unexpanded cells (median < 1.4 hPLT/uL). In contrast, hPLT were readily detected in 94±8% of mice injected with OMPC- and BS1-expanded cells (9- and 5-fold greater at day 4, and 26- and 32-fold greater at day 7, respectively; p<0.05 vs unexpanded and PBS controls). Conversely, hPLT levels in mice injected with unexpanded cells became and remained greater by 11 days PT (p<0.05). Finally, we investigated the impact of the length of expansion (6, 10 and 14-days) on the thrombopoietic potential of expanded CB cells. Mice transplanted with day-6, day-10 and day-14 BS1-expanded cells received an average dose of 1.3, 11.3 and 22.7 millions CD41+ Mks. Despite the increasing Mk cell doses, an inverse correlation between the length of expansion and hPLT levels were observed 14- (p<0.001) and 28-days PT (p<0.005). A similar correlation was observed for the number of huCD41+ cells in the BM 14-days PT (p<0.01). An inverse correlation between the length of culture and the total number of CD34+ and GPA+ human BM cells was also evident. As previously observed, hPLTs levels 14-days PT were the highest in mice transplanted with the unexpanded cells (p<0.002). High hPLT levels were detected in all cohorts up to 16 weeks PT, with the highest level observed in the unmanipulated and day-10 expanded groups.In summary, hPLT levels derived from unmanipulated CB cells were insignificant prior to 11 days PT, while hPLTs were readily detected with OMPC- and BS1- expanded CB cells. However, hPLTs production from unmanipulated cells became and remained dominant by 11–14 days PT. These results also confirm that cytokine cocktails promoting Mk-P expansion can improve short term platelet recovery and that expansion under MH is of added benefit for short term hPLT recovery.In conclusion, this work reveals that the hPLT reconstitution kinetics differ considerably between expanded and unmanipulated CB cells. Importantly, this difference support the concept that co-transplantation of both cell source could be of significant benefit for the short term platelet recovery in the context of CB transplantation. We are now in the process of evaluating this potential synergy. Disclosures:No relevant conflicts of interest to declare.

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

AbstractAbstract 3722Ex 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.

Expansion of Human Cord Blood Hematopoietic Stem Cells for Transplantation

A recent Science paper reported a purine derivative that expands human cord blood hematopoietic stem cells in culture (Boitano et al., 2010) by antagonizing the aryl hydrocarbon receptor. Major problems need to be overcome before ex vivo HSC expansion can be used clinically.

Substance P and calcitonin gene-related neuropeptides as novel growth factors for ex vivo expansion of cord blood CD34+ hematopoietic stem cells

There is little evidence on roles of growth factors other than cytokines in expansion of cord blood (CB) stem cells. We aimed to explore a novel approach for expansion, using Substance P (SP) and calcitonin gene-related peptide (CGRP) neuropeptides.CB CD34+ cells were cultured in different concentrations of SP and/or CGRP in combination with a cytokine cocktail. Phenotypic and functional analysis was performed by flowcytometry and colonogenic assay.Our results show a significant improvement of total expansion of neuropeptide treated cells. There was a selective effect of CGRP on CD34+ CD133+ cells, SP on CD34+ CD45dim cells, and 10− 9 M SP and/or CGRP on expansion of CD34+ CD38− cells. There was also a tendency for erythroid and granulocyte–myeloid colony formation in SP and CGRP treated cultures, respectively.Supplementation of cytokines with other growth factors, such as neuropeptides, might enable us to overcome the difficulties of ex vivo expansion of CB cells.

Possible Epigenetic Regulation of Functionally Opposing Groups of Genes Involved in Ex Vivo Expansion of Cord Blood Stem Cells

Recently, we have demonstrated that modifications of chromatin involving DNA methylation and histone acetylation events can change the fate of hematopoietic stem cells (HSC), likely as a result of altered gene expression patterns (Araki H et al. Blood 2007). In our current studies we have examined the epigenetic regulation of genes induced during the expansion of HSC by chromatin modifying agents [5aza-2-deoxycytidine (5azaD), trichostatin A (TSA)]. The expression of the polycomb repressing complex gene, enhancer of zeste homolog 2 (Ezh2) has been implicated in histone methylation and deacetylation. We observed a significant increase in the transcript levels of Ezh2 in addition to self-renewal genes HoxB4, Bmi1 and GATA-2 in 5azaD/TSA treated expanded human cord blood (CB) cells. Ezh2 overexpression has been shown to prevent HSC exhaustion in a murine model by chromatin stabilization. CB CD34+ cells expanded in 5azaD/TSA resulted in retention of relatively higher transcript levels of DNA methyltransferase (DNMT), DNMT1 and lower levels of DNMT3a and DNMT3b in CD34+CD90+ cells, the most primitive subpopulation, in contrast to the relatively mature CD34−CD90− cells. The retention of higher DNMT1, the maintenance methyltransferase, in the most primitive CD34+CD90+ cells may favor the chromosomal stability of expanded grafts. Cytogenetic analysis and DNA ploidy of expanded grafts were normal and the telomere length of expanded CB grafts did not reveal any significant alteration of their lengths in comparison to unmanipulated primary CB grafts. In order to explore the roles of epigenetic modifications on the differentiation of HSC during ex vivo expansion, we have also examined the transcript levels of genes associated with differentiation in 5azaD/TSA expanded cells in comparison to cells expanded in cytokines alone. Interestingly, the transcript levels of differentiation associated genes Pu.1 and GATA-1 decreased following 5azaD/TSA treatment. The lower transcript levels of differentiation inducing genes (Pu.1, GATA-1) and higher levels of self-renewing genes (Ezh2, HoxB4) in 5azaD/TSA expanded cells suggest a possible interaction among these functionally opposing groups of genes which are likely regulated by epigenetic mechanisms. By using a chromatin immunoprecipitation (ChIP) assay we observed significantly increased levels of histone H4 acetylation on the promoter sites of Hox-B4, Bmi-1, and GATA-2 and decreased levels of acetylation on the promoter sites of genes involved in differentiation (Pu.1, GATA-1) in 5azaD/TSA treated CB cells. The degree of histone H4 acetylation of these promoters sites directly correlated with the transcript levels of these genes as evidenced by real time quantitative PCR. Futhermore, silencing of HoxB4 by using short hairpin RNA (shRNA) during 5azaD/TSA mediated expansion resulted in about a 50% reduction in the expansion of CB cells in contrast to cells containing non-silencing control shRNA, indicating HoxB4 is required for HSC expansion. Understanding how the possible interactions between functionally opposing groups of genes regulating the expansion and differentiation of HSC is mediated by epigenetic mechanisms may further facilitate the optimization of ex vivo expansion strategies of CB grafts for therapeutic use.

Glycogen Synthase Kinase-3β Inhibition Preserves Hematopoietic Stem Cell Activity and Inhibits Leukemic Cell Growth

Ex vivo expansion of cord blood cells generally results in reduced stem cell activity in vivo. Glycogen synthase kinase-3beta (GSK-3beta) regulates the degradation of beta-catenin, a critical regulator of hematopoietic stem cells (HSCs). Here we show that GSK-3beta inhibition activates beta-catenin in cord blood CD34(+) cells and upregulates beta-catenin transcriptional targets c-myc and HoxB4, both known to regulate HSC self-renewal. GSK-3beta inhibition resulted in delayed ex vivo expansion of CD34(+) cells, yet enhanced the preservation of stem cell activity as tested in long-term culture with bone marrow stroma. Delayed cell cycling, reduced apoptosis, and increased adherence of hematopoietic progenitor cells to bone marrow stroma were observed in these long-term cultures treated with GSK-3beta inhibitor. This improved adherence to stroma was mediated via upregulation of CXCR4. In addition, GSK-3beta inhibition preserved severe combined immunodeficiency (SCID) repopulating cells as tested in the nonobese diabetic/SCID mouse model. Our data suggest the involvement of GSK-3beta inhibition in the preservation of HSC and their interaction with the bone marrow environment. Methods for the inhibition of GSK-3beta may be developed for clinical ex vivo expansion of HSC for transplantation. In addition, GSK-3beta inhibition suppressed leukemic cell growth via the induction of apoptosis mediated by the downregulation of survivin. Modulators of GSK-3beta may increase the range of novel drugs that specifically kill leukemic cells while sparing normal stem cells.

Characterization of Ex Vivo Expanded Cord Blood Graft Treated with Chromatin Modifying Agents for Potential Clinical Use.

The lack of optimal number of repopulating hematopoietic stem cells (HSC) and graft failure following transplantation remain as obstacles to using cord blood (CB) as a true alternative graft for adult patients. Using chromatin modifying agents {5aza-2-deoxycytidine (5azaD) and trichostatin A (TSA)}, we have developed a culture regimen that permits expansion of primitive CB CD34+ CD90+ cells resulting in a 10 fold expansion of in vivo repopulating cells without jeopardizing their multilineage differentiation capacity. Our results also indicate that the chromatin modifying agents likely mediate this function by inducing expression of endogenous genes (HoxB4, Bmi-1, GATA-2, etc.) previously implicated in self-renewal of HSC (Araki H et al. Blood 2007: 109:3670–3678). In our current studies we have further characterized the allostimulatory function of these expanded CB to assess its potential as grafts to mount an immune response. The percentage of 5azaD/TSA treated expanded CB cells co-expressing HLA-DR and CD86, or CD11c was less than cells expanded in cytokines alone. The expression of HLA-DR/CD40 was not altered following 5azaD/TSA treatment. In a mixed lymphocyte reaction (MLR), the irradiated CB cells (stimulator) were co-cultured with allogeneic peripheral blood T cells (responder) at 1:2 ratios. The MLR results indicate that 5azaD/TSA treated expanded CB grafts possessed diminished allostimulatory response as revealed by their relatively lower stimulation index in comparison to cells expanded in cytokines alone. Interestingly, the stimulation index of 5azaD/TSA treated cells (day 9) was even less than unmanipulated primary CB CD34+ cells (day 0). The reduced allostimulatory capacity of 5azaD/TSA expanded CB cells may favor in the reduction of incidence of graft rejection / graft failure following infusion. Alterations in cell fate regulatory mechanisms raise concerns for tumorigenesis, so the safety of such grafts was further assessed by examining the telomere length and cytogenetic analysis. Both cytogenetic analysis and examination of cell-cycle showed normal karyotype and diploid nature of expanded grafts, respectively. The telomere length of ex vivo expanded grafts as a putative indicator for their proliferative potential did not reveal significant alteration in their length in comparison to unmanipulated CB grafts. Also, in vivo repopulation assays with xenogeneic NOD/SCID mouse model showed no apparent tumor development following transplantation of ex vivo expanded 5azaD/TSA treated CB cells. In summary, our results indicate that the chromatin modifying agents not only result in expansion of in vivo repopulating CB cells but are capable of suppressing immunostimulatory capacity of expanded grafts and these grafts retain their telomere length and normal ploidy and can be expanded from a single unit for potential clinical use.