Fresh CD34 Research Articles

A three-step method for preparing cryopreserved samples of apheresis products for post-thaw analysis yields a high recovery of viable cells.

Flow cytometry protocols for counting fresh CD34+ cell samples are not ideal for cryopreserved products due to cryoprotectant cytotoxicity. For cryopreserved samples, often large volumes of hypotonic solutions, which can cause cell death, are used to remove the cryoprotectant with a post-thaw wash. We recently developed a novel multistep dilution method with subsequent flow cytometry analysis to allow for accurate and reproducible results. The previous method involved washing steps which invalidate the ability to enumerate cell recovery, and success had to be gauged solely on viability. The new method allows for assessment of total cell recovery and viable cell recovery. Apheresis products were cryopreserved in 10% DMSO at a target WBC concentration of 300 × 106/mL. Cryovials from these products were thawed at 37°C, and samples were diluted 1:2 by three additions of 1/3 sample volume using 1%-Human Albumin in Dextran 40 (10% Low Molecular Weight Dextran in 0.9% NaCl) separated by 5 min between each addition. A 1:10 dilution was performed to obtain the correct cell concentration for flow cytometric analysis resulting in a 1:20 dilution. End WBC concentrations were ~15 × 106/mL with DMSO diluted to 0.5%. Fifty-two samples were tested with this new method. Total and viable cell recoveries were calculated based on pre-cryopreservation data. Median total cell recoveries for CD34 and CD3 were >85%, while median viable cell recoveries were >75%. Cryopreserved samples can be reliably prepared for flow cytometric testing using a step-wise dilution to preserve cell integrity and robust recoveries.

One Year Follow-up on the First Patient Treated with Nula-Cel: An Autologous CRISPR/Cas9 Gene Corrected CD34+ Cell Product to Treat Sickle Cell Disease

Introduction Sickle cell disease (SCD) is the most common serious genetic disease in the world with over 500,000 births per year globally. It is estimated that there are >100,000 patients in the US with SCD and at least 20% have severe disease. Patients with SCD have at least one HBB gene with an adenine to thymidine variant at position 6 of the coding region resulting in a glutamic acid to valine amino acid change ( HbS). The success of allogeneic hematopoietic stem cell transplantation demonstrates that reconstitution of the hematopoietic compartment with HSCs that contain at least one non- HbS allele can cure the disease. Homology directed repair gene editing (HDR-editing) has achieved high frequencies of conversion of the thymidine to the non-pathologic adenine in CD34+ hematopoietic stem and progenitor cells from patients with SCD. Nulabeglogene autogedtemcel (nula-cel) is an investigational drug product in which HDR-editing of plerixafor-mobilized CD34+ HSPCs corrects the underlying variant that causes SCD. This is the first drug to directly correct a disease-causing variant. We report on the 1 year follow up of the first and only patient to receive nula-cel. Results Patient 1 is now a 23 year old female with homozygous SCD. She was consented and enrolled on a Phase I//II trial to test the safety and efficacy of nula-cel. In the 2 years prior to enrollment, the patient averaged 6 VOC's and 4 hospitalizations per year. Two aphereses of plerixafor-mobilized CD34+ cells were performed followed by fresh CD34+ cell purification. The purified CD34+ cells were pooled and cryopreserved. A 5-day manufacturing process starting with 9.3 x 10 6 cells/kg of cryopreserved CD34+ cells resulted in a yield of 8.75 x 10 6 CD34/kg with an on-target allele correction frequency of 33%. In August, 2022 the thawed drug product was infused after the patient received AUC-adjusted busulfan myeloablative conditioning chemotherapy. The initial cell viability was 77% giving a viable infused cell dose of 6.74 x 10 6 CD34/kg. Follow-up exploratory apoptois studies, however, showed the viable CD34+cell dose may have been as low as 3.5 x 10 6 CD34+/kg. Neutrophil engraftment occurred at transplant Day +40. Because of the lack of platelet recovery and continued platelet and RBC transfusion requirements, the patient was started on eltrombopag on day +106 (marrow cellularity of 5%). Since eltrombopag usage was not part of the protocol, its use prompted the reporting of an SAE but the trial was never put on FDA hold. The last dose of GCSF was D+140, the last platelet transfusion was D+181 and the last RBC transfusion was D+263. With rising platelet counts and hemoglobin and a stable ANC >1500, eltrombopag was discontinued on D+322. The blood counts have continued to slowly increase after discontinuation of the eltrombopag. On D+307, with a Hgb of 8.5 g/dl, hemoglobin electrophoresis demonstrated HgbA=12.5%, HgbF >78% and HgbS=4.5%. On D+349 the blood tests show Hgb=9.1 g/dl, Platelet=77,000/ul, ANC=1788/ul with an absolute reticulocyte count of 119,130/ul. There are no signs or symptoms of hemolysis. Table 1 shows the time course of gene marking with stable allele gene correction and INDEL frequencies since D+49. No change in off-target INDEL frequency nor change in on-target INDEL spectrum has occurred and no evidence of oligoclonal or clonal hematopoiesis has been observed. Clinically, the patient has shown marked improvement in quality of life with zero VOE's or other manifestations of SCD. The patient still has not reached a steady state and updated results will be presented. Conclusion We describe the clinical results of the first patient treated with an autologous cell product (nula-cel) in which the pathologic variant was directly corrected. The mechanism of action is fundamentally different than other clinical gene editing and gene therapy programs as the level of the pathologic HgbS is directly decreased thereby removing the dominant negative effect that HgbS has on red blood cells. The high HgbF is an unexpected but potentially effective mechanism of action for the clinical benefit and is being further studied. Longer follow up to assess the durability of the curative clinical impact is underway. Improvements in cell manufacturing are being implemented to shorten the duration of transfusion needs and increase the level of gene correction of engrafted cells. Plans to treat the next patients with an improved nula-cel product are in place.

Loss of Autophagy Activity during Storage of Hematopoietic Stem Cell Grafts Is Associated with Reduced Potency

Maximizing the potency of hematopoietic stem cell (HSC) grafts including cord blood (CB) units (CBU) is essential to minimize risk of engraftment delays and failures. Low cell dose in CB transplantation is partially responsible for the slower engraftment but processing delays could also be at play since CBU can be stored at room temperature (RT) up to 48 hours before cryopreservation. We hypothesized that such prolong storage reduces graft potencies due to a loss in hematopoietic stem cell and progenitors (HSPC) content. CBU were used as HSC graft. Units were split in 2 halves; one processed shortly after collection (<15 hrs, fresh) or after storage at RT (~43 hrs, stored). The impact of storage on graft quality was assessed using standard assays and bioinformatic analysis used to identify molecular pathways impacted by storage. Monitoring assays revealed that all CBU samples passed the minimal FACT-criteria threshold for post-thaw CD34+ cell viability and potency. However, the net number of ISHAGE CD34+ cell counts were reduced in stored samples (-20%, p<0.01). The colony forming-unit (CFU) assay used to measure the impact of storage on potency also revealed a 20% reduction in CFU in stored samples (<0.05, n=7). Losses in potency was confirmed with a second assay, the IL-3-phospho-STAT5 (pSTAT5) assay, which revealed a 10-fold loss in STAT5 signalling in stored vs. fresh CD34+ cells (p<0.0001, n=3). We hypothesized that factors released by CB could be responsible for the reduction of HSPC. In support of this, plasma isolated from stored CBU induced greater apoptosis of CD34+ cells than fresh plasma (+30% AnnexinV+ cells, p<0.01, n=3). This translated into losses of potency detectable after 30 minutes (CFU assay, p<0.001) and with further reduction over time peaking around 2 hours. Next, we sought to identify the molecular programs that govern this detrimental effect within CD34+ cells. Towards this we performed RNAseq analysis of CD34+ cells exposed to fresh or stored plasma for 20 minutes and 4 hours. A time course comparative analysis of differentially expressed genes (DEG, Log 2Fold >1.5, and q<0.05) between cells exposed to fresh vs stored plasma identified a total of 810 DEG genes. Gene ontology enrichment analysis identified autophagy, cell cycle, histone and DNA methylation and mRNA regulation as the major molecular programs that might be up regulated by the paracrine factors from fresh plasma but repressed by those from stored plasma. Autophagy flux assay confirmed that CD34+ cells isolated from stored UCB samples had reduced autophagy activity, and that stored plasma induced a 50% reduction in autophagy flux (p<0.001, n=3). Furthermore, RT-qPCR analysis confirmed that prolonged storage of CBU samples at RT increased the cellular senescence marker p21(CDKN1B), down regulated cell cycle genes ( CDK4 and CDK7) and autophagy genes ( ATG4, ATG12 and BECN1). We hypothesized that re-activation or prevention of autophagy loss could prevent losses of HSPC within CBUs stored at RT. To test this, CBUs were divided and individually supplemented with either autophagy activators (rapamycin or trehalose), autophagy inhibitor (3-methyladenine, 3-MA) or DMSO control and stored up to 43 hours. As expected, the early autophagy inhibitor 3-MA failed to restore HSPC numbers and potency. However, addition of trehalose prior to storage restored 99% of both net number and potency of HSPCs in HSC grafts as measured by the ISHAGE CD34+ counts, CFU and pSTAT5 assays to baseline level (n=3). Interestingly, the near complete restoration mediated by trehalose coincided with restauration of autophagy activity, repression of senescence gene CDKN1B and activation of cell cycle genes. In contrast, rapamycin only partially restored HSPC numbers and potency which coincided with the activation of autophagy genes but not others mentioned above. In conclusion, the loss of potency and viability seen in CBU grafts originates in part by paracrine-mediated mechanisms that lead to loss of autophagy, down regulation of cell cycle regulators and induction of senescence in HSPCs. Interestingly, addition of trehalose as a natural supplement precludes these molecular changes and restores CBU potency during storage. Taken together, these results stress the importance of rapid processing of HSC grafts and identify an attractive new solution to maintain high HSC graft potency post-collection during storage at ambient temperature.

Studies for Stability of Ex Vivo Produced Megakaryocytes and Their Distribution in Transplanted Mice

▪We previously established an efficient pilot-scale system to generate sufficient megakaryocytes/platelets for potential clinical applications. Here, we present recent studies that demonstrate the feasibility for stable preservation of ex vivo generated megakaryocytes/platelets (MKs/PLTs) and analyze distribution of these cells in transplanted NOD/SCID mice.The 8-day ex vivo culture of megakaryocyte progenitors (MKPs) were harvested and cryopreserved in liquid nitrogen for 1 or 3 months. Cells were then thawed and further cultured for 5 days so that these cells were differentiated toward the megakaryocytic lineage. Meanwhile, matured MKs/PLTs generated from fresh cord blood CD34+ cells on day 13 were collected and stored at 4°C with plasma substitution for 1, 3, 5, and 7 days, respectively. Subsequently, cell viability and specific bio-marker expression were analyzed for their phenotypic stability at various stored conditions. To observe distributions of ex vivo produced MKs/PLTs with transfected lenti-GFP in sub-lethally irradiated NOD/SCID mice (n=5) at the dose of 1.0×107 transfected cells/mouse. After transplantation for 0.5, 3, 6, and 24 h, mouse organs were harvested and images were immediately taken. Furthermore, peripheral blood of each mouse was collected and human GFP+CD41a+ MKs and GFP+CD42b+ PLTs were detected by flow cytometry to evaluate platelet-releasing potential of transplanted MKs in vivo . Mice transplanted with no GFP-labelled cells were used as negative control (n=3). Mice injected with saline alone were used as blank control (n=3).After 5 days' culture of MKPs that had been cryopreserved for 1 or 3 months, cell viability was 79.0%±3.3% or 75.2%±5.6%, with CD41a purity of 85.3%±4.5% or 87.2%±3.9% and CD61 purity of 68.4%±4.1% or 63.2%±2.3%, respectively. Cell viability and bio-marker expression of cryopreserved MKPs after in vitro differentiation were similar with 13-day culture of matured MKs/PLTs from freshly collected cord blood CD34+ cells (Viability: 85.5%±2.7%; CD41a: 82.4%±6.1% and CD61:74.0%±5.5%, respectively). For mature MKs/PLTs stored at 4°C, the cell viability of these four groups was 81.2%±4.5%, 74.3%±5.6%, 73.2%±4.9%, and 71.5%±6.3%, respectively; Percentages of CD41a cells of these four groups were 62.7%±5.1%, 58.3%±4.4%, 59.1%±6.0%, 56.9%±7.7% whereas percentages of CD61 cells were 65.8%±3.9%, 60.7%±2.5%, 45.2%±1.9%, 49.4%±4.4%, respectively. These results strongly suggest that MKPs of 8-day culture can be cryopreserved in liquid nitrogen for 3 months or longer, and matured MKs/PLTs of 13-day culture can be stored at 4°C for 7 days or longer without significantly affecting their viability and immunophenotypic characteristics. It is important to note that none of the groups had any bacterial and fungal contaminations before or after storage and during subsequent culturing processes. The fact that these cells were free of endotoxin and pyrogen strongly suggests that ex vivo generated MKs/PLTs are safe and stable. In mouse studies, no fluorescent signal could be detected in the blank control group, and only a trace amount of auto-fluorescence was detected in lung of the negative mouse group. However, in experimental group strong GFP signals could be observed in lung of each mouse 0.5 h after transplantation with the peak fluorescence between 3 and 6 h. Lung GFP signals remained detectable 24 h post transplantation. In mouse peripheral blood, human GFP+CD41a+ MKs could be detected only in the first 0.5 h after transplantation, and then human CD41a+CD42b+ PLTs emerged at 0.5 h with a low level of 0.5%±0.1%, after which the percentage increased rapidly with a peak level of 3.2%±0.6% at 6 h, indicating that the transplanted human MKs started to release PLTs after they merged into lung. Besides lung, a very weak signal was observed in the kidneys and livers, and completely disappeared at 24 h in mice of the experimental group. These results imply that engrafted mature MKs are capable of migrating into lung for further maturation, releasing platelets in NOD/SCID mice.MKPs of 8-day culture can be cryopreserved in liquid nitrogen for at least 3 months without affecting their viability and phenotypic characteristics. Mature MKs/PLTs of 13-day culture are stable in plasma substitute at 4°C for 7 days. The transplanted human MKs/PLTs appear to primarily home to the lung of NOD/SCID mice to release platelets in vivo . DisclosuresWang:Biopharmagen Corp.: Employment.

Universal Engraftment after Allogeneic Hematopoietic Cell Transplantation Using Cryopreserved CD34-Selected Grafts

As a result of the COVID-19 pandemic, most centers performing allogeneic hematopoietic cell transplantation (allo-HCT) have switched to the use of cryopreserved grafts. Previous investigators have suggested that cryopreserved allografts may heighten risk of nonengraftment. To date, no study has investigated the effect of cryopreservation of CD34-selected hematopoietic progenitor cells (CD34+ HPCs) used as the sole graft source. In this study, we sought to evaluate outcomes after unrelated donor or matched sibling allo-HCT with cryopreserved CD34+ HPCs. This was a single-center analysis of adult patients with hematologic malignancies who underwent allo-HCT with cryopreserved CD34-selected allo-HCT grafts between January 2010 and June 2017. All patients received ablative conditioning and antirejection prophylaxis with rabbit antithymocyte globulin. G-CSF-mobilized leukapheresis products underwent CD34 selection using the CliniMACS Reagent System. Cells were then cryopreserved in DMSO (final concentration 7.5%) to -90 °C using a controlled-rate freezing system before being transferred to vapor-phase liquid nitrogen storage. In internal validation, this method has shown 92% mean CD34+ cell viability and 99.7% mean CD34+ cell recovery. Engraftment was defined as the first of 3 consecutive days of an absolute neutrophil count of ≥0.5. Platelet recovery was recorded as the first of 7 consecutive days with a platelet count ≥20 K/μL without transfusion. Kaplan-Meier methodology was used to estimate overall survival (OS) and relapse-free survival (RFS), and cumulative incidence functions were used to estimate rates of relapse, nonrelapse mortality (NRM), and acute graft-versus-host disease (GVHD). A total of 64 patients received a cryopreserved CD34-selected graft. The median CD34+ cell count before cryopreservation was 6.6×106/kg (range, 1.4 to 16.1×106/kg), and the median CD3+ cell count was 2.0×103/kg (range, 0 to 21.1×106/kg). All patients were engrafted, at a median of 11 days post-HCT (range, 8 to 14 days). One patient had poor graft function in the setting of cytomegalovirus viremia, necessitating a CD34-selected boost on day +57. The median time to platelet recovery was 16 days (range, 13 to 99 days). The estimated 2-year OS was 70% (95% confidence interval [CI], 58% to 83%) with cryopreserved grafts versus 62% (95% CI, 57% to 67%) with fresh grafts (hazard ratio [HR], 0.86; 95% CI, 0.54 to 1.35; P=.5). The estimated 2-year RFS in the 2 groups was 59% (95% CI, 48% to 74%) versus 56% (95% CI, 51% to 61%; HR, 1.01; 95% CI, 0.68 to 1.51; P > .9). The cumulative incidence of relapse at 2 years was 29% (95% CI, 17% to 41%) versus 23% (95% CI, 19% to 27%; P=.16), and the cumulative incidence of NRM at 2 years was 17% (95% CI, 9% to 28%) versus 23% (95% CI, 19% to 28%; P=.24). The cumulative incidence of grade II-IV acute GVHD by day +100 was 16% with cryopreserved grafts (95% CI, 8% to 26%) and 16% (95% CI, 13% to 20%; P=.97) with fresh grafts. Moderate to severe chronic GVHD by day +365 occurred in only 1 recipient of a cryopreserved graft (2%). Our data show that in patients with hematologic malignancies who received cryopreserved allogeneic CD34+ HPCs, engraftment, GVHD, and survival outcomes were consistent with those seen in recipients of fresh allogeneic CD34+ HPC grafts at our center. Our laboratory validation and clinical experience demonstrate the safety of our cryopreservation procedure for CD34-selected allografts.

An Improved Patient-Derived Xenograft Humanized Mouse Model for Evaluation of Lung Cancer Immune Responses.

Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord blood CD34+ stem cells (CD34+ HSC), and combined it with lung cancer cell line-derived human xenografts or patient-derived xenografts (Hu-PDX). Fresh CD34+ HSCs could reconstitute detectable mature human leukocytes (hCD45+) in mice at four weeks without the onset of graft-versus-host disease (GVHD). Repopulated human T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) increased in peripheral blood, spleen, and bone marrow over time. Although cultured CD34+ HSCs labeled with luciferase could be detected in mice, the cultured HSCs did not develop into mature human immune cells by four weeks, unlike fresh CD34+ HSCs. Ex vivo, reconstituted T cells, obtained from the tumor-bearing humanized mice, secreted IFNγ upon treatment with phorbol myristate acetate (PMA) or exposure to human A549 lung tumor cells and mediated antigen-specific CTL responses, indicating functional activity. Growth of engrafted PDXs and tumor xenografts was not dependent on the human leukocyte antigen status of the donor. Treatment with the anti-PD-1 checkpoint inhibitors pembrolizumab or nivolumab inhibited tumor growth in humanized mice significantly, and correlated with an increased number of CTLs and decreased MDSCs, regardless of the donor HLA type. In conclusion, fresh CD34+HSCs are more effective than their expanded counterparts in humanizing mice, and do so in a shorter time. The Hu-PDX model provides an improved platform for evaluation of immunotherapy.

Abstract 4984: Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in lung cancer

Abstract Current preclinical models of non-small cell lung cancer (NSCLC) do not recapitulate the human tumor microenvironment. Mice reconstituted with a human immune system and bearing human patient derived xenografts may be advantageous in evaluating human anti-tumor immune response. We developed an improved NOD scid gamma (NSG) mouse model derived from non-expanded CD34+ stem cells, without CD3+ T cell contamination, to evaluate antitumor responses to immunotherapy in NSCLC. Using fresh CD34+ from umbilical cord blood reduced humanization time significantly. Human CD45+ cell reconstitution with increased functional human lymphoid (B, T, monocytes and NK cells) and myeloid (macrophages and MDSCs) lineage repopulation, without the onset of GvHD, was achieved as early as 4 weeks post-stem cell engraftment. Published studies using expanded CD34+ derived humanization reveal compromised purity of CD34+ stem cells with an increasing number of mononuclear cells. Reconstitution of CD8+ and CD4+T cells is not achieved until 12 to 15 weeks post-engraftment at much lower levels than fresh CD34+ humanization. Single cell suspension analysis shows levels of human reconstituted T, B, NK, DC and MDSC cells at 4 weeks, which increased significantly at 6 and 9 weeks in peripheral blood, spleen and bone marrow. Human repopulated T cells were functionally active in secretion of IFN-γ by mitogenic stimuli such as PMA and IL-2 and by allogenic human cancer cells. Antigen specific CTL responses were observed when reconstituted human T cells from PDX bearing humanized mice were challenged with PDX tumor. No non-antigen specific responses were observed when T cells were co-cultured with HLA-matched human bronchial epithelial cells (HBEC). To evaluate the applicability of the humanized mouse in lung cancer translational research, we combined it with Hu-PDX or Hu-xenograft tumors and analyzed tumor growth and treatment response to the anti-PD1 checkpoint inhibitor pembrolizumab. We found that efficient engraftment of PDXs and xenograft tumors were not dependent on donor HLA-status. Similar to the clinical outcome, treatment with pembrolizumab, inhibited tumor growth significantly in both Hu-PDX, and Hu-xenograft mice regardless of donor HLA-types, increasing cytotoxic T cells and decreasing MDSC levels. Pembrolizumab had no effect on the non-humanized NSG controls. In concordance with our previous study with a syngeneic mouse tumor, the antitumor effect of check point blockade was significantly enhanced when combined with nanoparticle systemically deliveredTUSC2, a tumor suppressor and immunomodulatory gene, in a KRAS mutant lung metastasis humanized mouse model. In conclusion, fresh CD34+ are more effective than their expanded counterparts in humanizing mice, do so in much reduced time, and recapitulate the immune response to cancer. Citation Format: Ismail M. Meraz, Mourad Majidi, Feng Meng, RuPing Shao, Min Jin Ha, Shinya Neri, Bingliang Fang, Steven H. Lin, Peggy T. Tinkey, Elizabeth J. Shpall, Jeffrey Morris, Jack A. Roth. Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4984.

Cryopreservation and thawing of hematopoietic stem cell CD34-induced apoptosis through caspase pathway activation: Key role of granulocytes

IntroductionCell damage inescapably occurs during both the freezing and the thawing graft processes for autologous hematopoietic stem cell (HSC) transplantation. To estimate HSC injury, a quality control is performed including: (i) CD34+ quantification; (ii) percentage of CD34+ viability and (iii) evaluation of HSC functional ability to form colony forming unit–granulocyte macrophage (CFU-GM). Apoptosis involves complex pathways such as caspase enzymes. Here, we assess the extent of apoptosis that is caspase-dependent before and after cryoconservation of CD34+, using a Fluorescent Labeled Inhibitor of CAspases (FLICA). MethodsCaspase pathway activation status was evaluated in 46 patients (multiple myeloma [n = 24], lymphoma [n = 22]), by flow cytometry, using a 7-aminoactinomycin-D (7AAD)/FLICA staining test, in CD34+, CD3+, CD14+ and CD56+ cells. Viable 7AAD−/FLICA+ cells were then correlated with various parameters. ResultsWe showed a significant caspase pathway activation, with 23% CD34+/7AAD−/FLICA+ cells after thawing, compared with the 2% described in fresh CD34+ cells (P < 0.0001). Moreover, caspase pathway was significantly activated in thawing CD3+, CD56+ and CD14+ cells. We also report a significant correlation between the rate of CD34+/7AAD−/FLICA+ cells and post-thawing granulocytes count (P = 0.042) and their potential to be differentiated into CFU-GM (P = 0.004). DiscussionOur results show substantial cell death, induced by the increase of caspase pathway activation, secondary to the thawing process, and across all study cell types. This observation may affect the immune response quality during recipient aplasia, without detecting a clinical impact. Moreover, caspase pathway activation through CD3+ and CD56+ subpopulations could modify the therapeutic result of donor lymphocytes infusion (DLI).

Advances in the Gene Therapy of Patients with Fanconi Anemia

▪Fanconi anemia (FA) is a DNA repair syndrome characterized by bone marrow failure, congenital abnormalities and cancer predisposition. Based on previous experimental results showing the in vivo proliferative advantage of gene corrected FA patients' hematopoietic stem cells (HSCs; Rio, Navarro et al. Blood 2017) a gene therapy trial in non-conditioned FA-A patients was initiated in 2016. Six patients have been treated to-date using fresh and cryopreserved CD34+ cells mobilized to peripheral blood with G-CSF and plerixafor, and transduced with the PGK-FANCA.Wpre* lentiviral vector. Cell doses infused in four patients with a follow-up of at least 12 months varied from 0.6 to 1.4 million CD34+ cells/kg. Transduction efficacies of these samples, determined as vector copies per cell, ranged from 0.17 to 0.53 copies/cell. Despite the absence of patients' conditioning, a marked in vivo expansion of gene-corrected cells was observed in all hematopoietic cell lineages analyzed in BM and PB. Significantly, up to 44% of corrected cells were determined in total PB cells at the most recent follow-up visit (24 month) in the first treated patient. Insertion site analyses in PB cells showed an oligoclonal pattern of hematopoietic reconstitution, and revealed engraftment of multipotent corrected HSCs and no evidence of insertion-site mediated clonal expansion. Functional studies showed significant increases in the resistance of BM progenitors to mitomycin C in all treated patients. Additionally, patients with higher levels of corrected cells also showed significant increases in the chromosomal stability of T cells exposed to diepoxybutane. Finally, analyses discriminating the presence of corrected and uncorrected PB cells in these patients showed marked increases in the total number of corrected leukocytes, contrasting to progressive decreases of uncorrected cells. Our studies demonstrate for the first time that lentiviral-mediated gene therapy results in progressive engraftment and phenotypic correction of HSCs in non-conditioned FA patients, suggesting that this gene therapy approach may constitute a low-toxicity option for the treatment and prevention of BMF in patients with FA. DisclosuresBueren:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Casado:Rocket Pharmaceuticals Inc: Patents & Royalties. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH: Employment; German Cancer Research Center: Employment; bluebird bio: Consultancy. Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals Inc: Honoraria, Patents & Royalties.

The Functional Effect of Repeated Cryopreservation on Transduced CD34+ Cells from Patients with Thalassemia

Stable gene marking and effective engraftment of gene-modified CD34+ hematopoietic stem cells is a prerequisite for gene therapy success but may be challenged by the inevitable cryopreservation of the final product prior to extensive quality assurance testing. We investigated the β-globin gene transfer potency in fresh and cryopreserved CD34+ cells from mobilized patients with β-thalassemia, as well as the qualitative impact of repeated freeze/thaw cycles on the functionality of cultured and unmanipulated CD34+ cells in terms of engrafting capacity in a xenotransplantation model, under partial myeloablation. Cells transduced fresh or after one freeze-thaw cycle yielded similar clonogenic and gene transfer frequencies. Repeated cryopreservation cycles did not affect the transduction rates whereas either one or two freeze-thaw cycles of cultured-but not of unmanipulated-cells significantly reduced their clonogenicity. No differences in the engrafting potential of gene-corrected cells subjected to either none or up to two cryopreservation cycles, were encountered post xenotransplantation. Overall, we assessed the gene transfer efficiency, clonogenicity and engrafting capacity of cryopreserved CD34+ cells and the impact of repeated freeze/thaw cycles in their performance. These observations may prove essential in the design of gene therapy trials, considerably facilitating their logistics.

Fresh or Cryopreserved CD34+-Selected Mobilized Peripheral Blood Stem and Progenitor Cells for the Treatment of Poor Graft Function after Allogeneic Hematopoietic Cell Transplantation

CD34+-selected stem cell boost (SCB) without conditioning has recently been utilized for poor graft function (PGF) after allogeneic hematopoietic stem cell transplantation with promising results. Unfortunately, many patients have been unable to receive the boost infusion as their donors were unwilling or unable to undergo an additional stem cell collection. Therefore, we conducted this study utilizing either fresh or cryopreserved peripheral blood stem cell products to create CD34+-selected boost infusions for the treatment of PGF. Additionally, to explore relationship of CD34+ dose and response, we included a cohort of donors mobilized with plerixafor in addition to the standard granulocyte colony–stimulating factor (G-CSF). Twenty-six patients with PGF were included in this study. Seventeen donor-recipient pairs were enrolled onto the prospective study; an additional 9 patients treated off protocol were reviewed retrospectively. Three different donor products were used for CD34+ selection: (1) fresh mobilized product using G-CSF only, (2) fresh mobilized products using G-CSF and plerixafor, and (3) cryopreserved cells mobilized with G-CSF. CD34+ cell selection was performed using a CliniMACS. The infusion was not preceded by administration of any chemotherapy or conditioning regimen. The primary objective was hematologic response rate and secondary objectives included CD34+ yields, incidence and severity of acute and chronic graft-versus-host disease (GVHD), overall survival (OS), and relapse-free survival (RFS). The median post-selection CD34+ counts per kilogram of recipient weight were 3.1 × 106, 10.9 × 106, and 1 × 106 for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. The median CD34+ yields (defined as the number of CD34+ cells after selection/CD34+ cells before CD34+ selection) were 69%, 66%, and 28% for G-CSF only, G-CSF plus plerixafor, and cryopreserved products, respectively. After SCB, 16 of the 26 recipients (62%) had a complete response, including 5 of 8 (63%) who received cryopreserved products. Five had a partial response (19%), resulting in an overall response rate of 81%. One-year RFS and OS were 50% and 65%, respectively. There was no treatment-related toxicity reported other than GVHD: 6 (23%) developed acute GVHD (2 grade I and 4 grade II) and 8 (31%) developed chronic GVHD (2 limited and 6 extensive). Cryopreserved products are viable alternatives to create SCB for the treatment of PGF. When collecting fresh products is an option, the addition of plerixafor increases CD34+ yield over G-CSF alone; however, it is currently unclear if the CD34+ cell dose impacts the efficacy of the SCB.

Single-Cell RNA-Seq Reveals the Differentiation Hierarchy of Normal Human Bone Marrow and a Distinct Transcriptome Signature of Monosomy 7 Cells

Monosomy 7 is a frequent cytogenetic abnormality in hematopoietic malignancies and a general indicator of poor prognosis. Due to lack of distinct cell surface markers between monosomy 7 cells and normal cells, it is not feasible to physically separate aneuploid from diploid cells. We performed single-cell RNA-seq (scRNA-seq), which allows the entire transcriptome of large numbers of single cells to be assayed in an unbiased way, to investigate hematopoietic differentiation of normal and aneuploid human hematopoietic cells. Bone marrow samples were collected from four patients (P1-P4) with myelodysplastic syndrome, and four healthy volunteers. Conventional cytogenetics showed -7/7q- in bone marrow cells from P1, P3 and P4, and dup(1)(q21q32) in cells from P2; retrospectively, P2 was found positive for monosomy 7 as well as trisomy 8 by fluorescence in situ hybridization. Fresh CD34+CD38- and CD34+CD38+cells were sorted by flow cytometry and then subjected to Fluidigm C1 Single-Cell Auto Prep System for scRNA-seq. After excluding cells with low transcriptome coverage, 326 cells from P1 and P2 (analysis is in progress for P3 and P4), and 391 cells from healthy subjects were analyzed by comparison of transcriptomes from 17,071 genes. Nonlinear dimension reduction and visualization were achieved using t-distributed Stochastic Neighbor Embedding (tSNE). Cells from healthy controls clustered into seven subgroups based on their gene expression pattern, and each group could be associated with a previously reported hematopoietic cell type by known marker genes (Laurenti E, Nat Immunol, 2013). These cell types included hematopoietic stem cell (HSC), multilymphoid progenitor (MLP), granulocyte-monocyte progenitor (GMP), Pro-B cell (ProB), earliest thymic progenitor (ETP), and megakaryocytic-erythroid progenitor (MEP) (Fig 1a). Individual cells from healthy controls were ordered by Monocle software based on their expression profile similarity to uncover a differentiation hierarchy. A two-branch trajectory of development from HSC was revealed, with one branch progressing towards erythroid cell and the other to lymphoid/myeloid cells (Fig 1b). This pattern differs from the classic hematopoietic model, but is consistent with reports claiming existence of early-lymphoid-biased progenitors that retain myeloid but not erythroid potential (Doulatov S, Nat Immunol, 2010), and of dominance of multipotent and unipotent progenitors over scarce oligopotent progenitors in the adult marrow hematopoietic hierarchy (Notta F, Science, 2016). We compared single cells from patients and healthy controls for regional and chromosomal copy number differences in gene expression. We identified subclonal populations of cells from patients that showed decreased expression of chromosome 7 genes (60% in P1, and 55% in P2; Fig 1c and 1d), and increased expression of chromosome 8 (77% in P2) and chromosome 1 long arm genes (P2), at FDR=0.05 estimated with cells from health donors. Gene Ontology enrichment analysis using topGO indicated that cells with low global expression of chromosome 7 genes had dysregulated expression of immune related genes, including B cell receptor signaling pathway, T cell activation and differentiation, antigen receptor-mediated signaling pathway, as well as signal transduction and Fc-γ Receptor signaling pathway. ScRNA-seq analysis reveals a simple pattern of normal human hematopoietic development and the molecular signature of aneuploid cells from patients with developing "clonal evolution". This powerful method should improve characterization of functional changes in human cells with chromosome abnormalities. [Display omitted] DisclosuresDesierto:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding. Young:Novartis: Research Funding.

Human Umbilical Cord Blood CD34+Cells InducedAngiogenesis in Ischemic Limb of Mice.

Objective To observe the effect of the expanded human umbilical cord blood CD34+cells in ischemic limb of mice and analyse the relationship between the CD34+cells and angiogenesis. Methods Human umbilical cord blood was collected and CD34+cells were separated for expanding. Mice limbs ischemia models were established (n=15) and randomly divided into three groups:expanded CD34+cells group (n=5),fresh CD34+cells group (n=5),and control group(n=5). CD34+cells were detected by DiI dye tracing and antihuman nuclear antigen antibody(HNA) immunohistochemical staining. The improvement of blood reperfusion was evaluated by indicators including limb temperature,CD31 staining,and transforming growth factor-β1 (TGF-β1) mRNA expression. Results On days 14 (t=5.421,P=0.001;t=0.616,P=0.000) and 28(t=10.780,P=0.000; t=12.123,P=0.000),both expanded CD34+cells group and fresh CD34+cells group enjoyed better temperature improvement. Days 28 later,the vascular densities in the expanded CD34+cells group and the fresh CD34+cells group were 592.3±24.6 (t=26.386,P=0.000) and 530.7±25.5 (t=21.502,P=0.000),which were significantly higher than that in control group 219.7±19.9. The TGF-β1 mRNA expression in the expanded CD34+cells group and the fresh CD34+cells group were (0.578±0.050) copies (t=12.376,P=0.000) and (0.504±0.080) copies (t=7.098,P=0.000),both over control group [(0.224±0.040)copies]. Conclusions In vitro culture of cord blood CD34+cells can emigrate to ischemic zone and induce angiogenesis to alleviate ischemia. Thus,it may provide a treatment option for lower limb ischemia.

Cord Blood-Derived Quiescent CD34+ Cells Are More Transcriptionally Matched to AML Blasts Than Cytokine-Induced Normal Human Hematopoietic CD34+ Cells.

Acute myeloid leukemia (AML) is characterized by developmental arrest, which is thought to arise from transcriptional dysregulation of myeloid development programs. Hematopoietic stem and progenitor cells (HSPCs) isolated from human blood are frequently used as a normal comparator in AML studies. Previous studies have reported changes in the transcriptional program of genes involved in proliferation, differentiation, apoptosis, and homing when HSPCs were expanded ex vivo. The intrinsic functional differences between quiescent and dividing CD34+ HSPCs prompted us to determine whether fresh or cytokine-induced cord blood-derived CD34+ HSPCs are a more appropriate normal control compared to AML blasts. Based on principal component analysis and gene expression profiling we demonstrate that CD34+ HSPCs that do not undergo ex vivo expansion are transcriptionally similar to minimally differentiated AML blasts. This was confirmed by comparing the cell cycle status of the AML blasts and the HSPCs. We suggest that freshly isolated CD34+ HSPCs that do not undergo ex vivo expansion would serve as a better control to identify novel transcriptional targets in the AML blast population.

Apoptosis, DAP-Kinase1 Expression and the Influences of Cytokine Milieu and Mesenchymal Stromal Cells on Ex Vivo Expansion of Umbilical Cord Blood-Derived Hematopoietic Stem Cells

Expansion of umbilical cord blood-derived CD34(+) cells can potentially provide them in numbers sufficient for clinical applications in adult humans. In this study apoptosis rate of expanded cells, mRNA expression and promoter methylation status of DAPK1 were evaluated during cord blood hematopoietic stem cell (CB-HSC) ex vivo expansion using cytokines and a co-culture system with mesenchymal stromal cells (MSCs). Ex vivo cultures of CB-HSCs were performed in three culture conditions for 14days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer and co-culture with MSCs feeder layer without cytokine. Total number of cells, CD34(+) cells and colony forming unit assay were performed during expansion. Flow cytometric analysis by propidium iodide was performed to detection of apoptosis rate in expanded cells. Methylation status of the DAPK1 gene promoter was analyzed using methylation specific PCR, and DAPK1 mRNA expression was evaluated by real time-PCR. Maximum CB-CD34(+) cells expansion was observed in day 10 of expansion. The highest apoptosis rate was observed in cytokine culture without feeder layer that showed significant difference with co-culture condition. The data showed that ex vivo expansion of CD34(+) cells in all three culture conditions after 10days resulted in, significant increased expression of DAPK1, also a significant difference between co-culture without cytokine and two other cytokine culture was observed (p<0.01). DAPK1gene promoter of expanded CD34(+) cells at days 5, 10 and 14 of culture remained in unmethylated form similar to fresh CD34(+) cells. Expression of DAPK1 in hematopoietic cells was increased during 10days expansion of CD34(+) cells. Also no methylation of DAPK1 promoter was observed; otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Cryopreservation and Thawing of Hematopoietic Stem Cells CD34 Induced Apoptosis through Caspases Pathway Activation

BACKGROUD: Cryopreservation, before storage, is an essential step for autologous hematopoietic stem cells transplantation. Currently, the quality of the graft is estimated: by the quantification of CD34 cells after thawing, the percentage of viability of CD34+ cells evaluated by an Annexin V/7AAD staining and a functional CFU-GM assay. The cryopreservation protocols can affect the viability of HSC CD34+ after thawing which could affect the quality of the graft after injection. The cell death could be induced by cells necrosis or apoptosis. Apoptosis, or programmed cell death, involves complex pathways in part Fas ligand (FasL), mitochondrial components, caspases, cathepsins, and calpain. The involvement of apoptosis dependent on caspases activation pathway in the CD34+ quality has not been evaluated. We have conducted a study to assess the extent of apoptosis before and after cryopreservation of PBSC CD34+ cells using a Fluorescent Labelled Inhibitor of Caspases FLICA. We determined the percentage of FLICA positive cells in viable cells (7AAD negative cells) before and after thawing and confronted results to colony forming units potential and to the number of days of clinical aplasia.METHODS: we tested the induction of caspases-dependent apoptosis before and after cryoconservation in HSC CD34 cells from patients with different hematological malignances: multiple myeloma (n=11), Hodgkin’s (n=2) and non- Hodgkin’s lymphoma NHL (n=5), and amylose (n=1). We evaluated the caspases activation by flow cytometry using the Fluorescent Labelled Inhibitor of Caspases FLICA staining test. Caspases activation status was evaluated in lymphocytes CD3+, monocytes CD14+ and natural killer NK CD56+ cells. Cells were stained in parallel with 7AAD to visualize late dead cells. The correlation with colony forming units CFU-GM and the number of days of aplasia were evaluated.RESULTS: Caspases-dependent pathway is activated in 22% in CD34 cryoconseved after thawing while this percent was less than 5% in fresh CD34 (p<0.0001). Caspases activation was observed at significantly amounts in other thawing blood cells. Percent of FLICA thawing CD3+, CD56+ and CD14+ cells was 12, 25 and 5% compared to 2, 8 and less than 1% in fresh cells.Induction of caspase-dependent apoptosis in CD34+ after thawing affects clonogenicity. FLICA +CD34 reduced the clonogenicity. Only Flica negative cells are able to differentiate in CFU-GM. Length of aplasia was not affected by the percentage of CD34+ cells engage in a caspases-dependent apoptosis after thawing.CONCLUSION: Our results suggest that cryopreservation induced apoptosis by a caspases-dependent pathway in all the subset of apheresis product and mainly CD34 cells. These results modify the quality control of the product but do not affect the clinical outcome. The percent of CD3 and CD56 FLICA positive cells could modify the effect of DLI. More data will be presented DisclosuresNo relevant conflicts of interest to declare.

Expression and promoter methylation changes of the P15INK4b during ex vivo cord blood CD34+ cell expansion following co-culture with mesenchymal stromal cells

BackgroundBecause of the insufficient number of cord blood hematopoietic stem cells (CB-HSC), expansion of these cells seems to be important for clinical application in adults. Cell cycle inhibitors are important regulators in normal hematopoietic regeneration. In this study, mRNA expression and promoter methylation status of p15INK4b were evaluated during CB-HSC ex vivo expansion using cytokines and in co-culture system with a mesenchymal stem cells (MSCs) feeder layer.MethodsEx vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with an MSCs feeder layer, cytokines without a MSCs feeder layer, and co-culture with MSCs without cytokines. After expansion, measuring the total number of cells, CD34+ cells, and CFU assay was performed. Methylation status of the p15INK4b gene promoter was analyzed using methylation-specific polymerase chain reaction and p15 mRNA expression was evaluated by real-time reverse transcriptase polymerase chain reaction.ResultsMaximum CB-HSC expansion was observed on day 10 of expansion. The data showed that after 10 days, p15 mRNA expression in the expanded cells in all the three culture conditions was higher than in CD34+ fresh cells (P < 0.01). p15 gene promoter of expanded CD34+ cells remained in an unmethylated form just like fresh CD34+ cells in all the three culture conditions at days 5, 10, and 14 of culture.ConclusionsExpression of p15INK4b in HSCs was not decreased during ex vivo expansion. Also, no methylation of p15 promoter was observed, otherwise it would be capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.

Local Transplantation of Ex Vivo Expanded Bone Marrow-Derived CD34-Positive Cells Accelerates Fracture Healing

Transplantation of bone marrow (BM) CD34(+) cells, an endothelial/hematopoietic progenitor-enriched cell population, has shown therapeutic efficiency in the treatment of ischemic diseases enhancing neovascularization. However, the number of CD34(+) cells obtained from bone marrow is not sufficient for routine clinical application. To overcome this issue, we developed a more efficient and clinically applicable CD34(+) cell expansion method. Seven-day ex vivo expansion culture of BM CD34(+) cells with a cocktail of five growth factors containing VEGF, SCF, IL-6, Flt-3 ligand, and TPO resulted in reproducible more than 20-fold increase in cell number. The favorable effect of the local transplantation of culture expanded (cEx)-BM CD34(+) cells on rat unhealing fractures was equivalent or higher than that of nonexpanded (fresh) BM CD34(+) cells exhibiting sufficient therapeutic outcome with frequent vasculogenic/osteogenic differentiation of transplanted cEx-BM CD34(+) cells and fresh BM CD34(+) cells as well as intrinsic enhancement of angiogenesis/osteogenesis at the treated fracture sites. Specifically, cEx-BM CD34(+) cell treatment demonstrated the best blood flow recovery at fracture sites compared with the nonexpanded BM CD34(+) cells. In vitro, cEx-BM CD34(+) cells showed higher colony/tube-forming capacity than nonexpanded BM CD34(+) cells. Both cells demonstrated differentiation potential into osteoblasts. Since fresh BM CD34(+) cells can be easily collected from fracture sites at the time of primary operation and stored for future use, autologous cEx-BM CD34(+) cell transplantation would be not only a simple but also a promising therapeutic strategy for unhealing fractures in the field of orthopedic trauma surgery.

Repairing of Homing Defect in Cord Blood Hematopoietic Stem Cell Transplantation–Comparison of Fucosyltransferase VII with Fucosyltransferase VI.

AbstractAbstract 2988 Objective:Relatively slower speed of engraftment in bone marrow of recipients makes cord blood (CB) transplantation (CBT) more difficult in a clinical setting. Selectins and their ligands are known to play a role in the homing of hematopoietic stem/progenitor cell (HSPC) to the bone marrow. Fucosyltransferase VI (FTVI) mediated ex vivo fucosylation improves human CB engraftment in NOD-SCID IL-2Rγnull (NSG) mice. FTVI is expressed in many cell types, while fucosyltransferase VII (FTVII) is primarily expressed in hematopoietic cells. FTVII deficient leukocytes do not bind the P- and E-selectins and are not recruited to sites of inflammation. We hypothesize that FTVII is more efficient than FTVI in surface fucosylation of CB-HSPC and thus enhances homing and engraftment of CB-HSPC (CD34+) in NSG mice. Method:Purified CB CD34+ cells were obtained from fresh CB in healthy full-term newborns by immunomagnetic microbeads sorting. CD34+ cells were treated with FTVI or FTVII respectively, the expression level of sLex (HECA-452) and binding to P- or E-selectin were detected by flow cytometry. Interactions of FTVI-, FTVII- or combined FTVI/FTVII-fucosylated CB CD34+cells with P- or E-selectin were measured under physiological flow conditions using a parallel-plate flow chamber assay. CBT was performed in NSG mice of 5 groups (negative control, sham-control, FTVI-treated, FTVII-treated, and FTVI/FTVII-treated). From 2 weeks post transplantation, mice were bled every 2 weeks for anti-human CD45 screening by flow cytometry. We used this method to evaluate the engraftment progress of CB-HSPC with different treatments. Ten weeks after transplantation, all mice were sacrificed and peripheral blood and bone marrow cells were isolated. Hematopoiesis reconstitution of CB cells was analyzed by flow cytometry after staining the cells with antibodies to various human hematopoietic subset markers, which included CD45 for total leukocytes, CD19 for B-cells, CD33 for myeloid cells, CD41 for megakaryocytes, CD3 for T-cells, and CD34 for HSPCs. Result:Under in vitro conditions, surface fucosylation with FTVI or FTVII results in an enhanced expression of HECA-452 epitopes on CD34+ cells from fresh CB. Increased P- and E-selectin binding to fresh CB CD34+ cells after either FTVI or FTVII treatment compared to sham-treated. However, FTVII-treated fresh CB CD34+ cells had a much higher binding to P-selectin than that of FTVI-treated fresh CB CD34+ cells. FTVI/FTVII-fucosylated CD34+ cells isolated from fresh CB accumulated more on P- or E-selectin coated plates than that of single enzyme fucosylated CD34+ cells under flow conditions. Although it is not statistically significant, more FTVII-treated fresh CB CD34+ cells rolled on both P- and E-selectin surface than FTVI-treated fresh CB CD34+ cells did. FTVII- or FTVI/FTVII-treated CD34+cells from fresh CB engrafted significantly better in NSG mice than the FTVI-treated cells at 4 and 6 weeks. Ten weeks after transplantation, the bone marrows of mice that received FTVII-treated cells had significantly improved engraftment of human hematopoietic progenitors of some lineages (CD3, CD33, and CD41) compared to that of mice that received FTVI-treated cells. Conclusion:FTVII-treated CB-HSPCs have greater engraftment progress during early time after transplantation compared to FTVI. FTVII mediated fucosylation did not affect self-renewal of CB-HSPC. Hematopoiesis reconstitution of T-lymphocytes, myeloid cells, and megakaryocytes of FTVII is better than FTVI. Finally, FTVI and FTVII may have a cooperative effect on CB-HSPC homing to bone marrow. Disclosures:Miyaji:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Sato:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Miller:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Xia:Kyowa Hakko Kirin Co., Ltd.: Research Funding.

Expression of P16 cell cycle inhibitor in human cord blood CD34+ expanded cells following co-culture with bone marrow-derived mesenchymal stem cells

BackgroundBecause of insufficient number of cord blood hematopoietic stem cells (CB-HSC), expansion of these cells seems to be important for clinical application in adults. Cell cycle inhibitors are important regulators in normal hematopoietic regeneration. In this study, mRNA expression and promoter methylation status of p16 were evaluated during CB-HSC ex vivo expansion using cytokines and a co-culture system with mesenchymal stem cells (MSCs) feeder layer.MethodsEx vivo cultures of CB-HSCs were performed in three culture conditions for 14 days: cytokines with MSCs feeder layer, cytokines without MSCs feeder layer, and co-culture with MSCs without cytokine. After expansion, measuring total number of cells, CD34+ cells and colony-forming unit (CFU) assay was performed. Methylation status of the p16INK4a gene promoter was analyzed using methylation-specific polymerase chain reaction (PCR), and p16 mRNA expression was evaluated by real-time reverse transcriptase-PCR.ResultsMaximum CB-HSC expansion was observed in day 10 of expansion. The data showed that after 10 days, p16 mRNA expression in the expanded cells at the co-culture system without cytokine was higher than in CD34+ fresh cells (P < 0.01); however, p16 mRNA expression in the expanded cells at both cytokine cultures with and without MSCs feeder layer was decreased. p16 gene promoter of expanded CD34+ cells remained in unmethylated form just like fresh CD34+ cells in all the three culture conditions at days 5, 10, and 14 of culture.ConclusionExpression in HSCs of p16INK4a, an important cell cycle regulator in normal hematopoietic regeneration disruption of which is involved in leukemic cell development, was increased during 10 days of expansion in co-culture with MSCs feeder layers. Also, no methylation of p16 promoter was observed, which is capable of initiating some leukemic cell progression or disruption in hematopoietic regeneration.