Expanded Cell Product Research Articles

Enhanced stability of cell-based products with recombinant human serum albumin in combination with an optimized inclusion of cryoprotective agents in a chemically defined cryoprotective media

Background & Aim The need for a stable final cell product to be delivered to the patient is essential to ensure product efficacy and safety with cellular therapies. It is well known that human serum albumin has critical cryoprotective activity for cell-based products in final formulations that are to be administered to the patient. However, use of plasma-derived albumin can introduce inconsistency through raw material variability which can negatively impact the final cell product efficacy and reproducibility. Recombinant human serum albumin offers an attractive solution to circumvent these challenges that arise from raw material heterogeneity in manufacturing and distribution of cell-based products. Methods, Results & Conclusion Here, we have incorporated a novel recombinant human serum albumin into an optimized chemically defined and blood-free cryopreservation media to enhance the stability of previously expanded T Lymphocytes. Cell viability was measured at multiple time points post-thaw to ensure prolonged stability of the expanded cell product. Cells cryopreserved in the chemically defined media maintained viability over time that was comparable to current industry standard final formulations. Phenotype of the cryopreserved T Lymphocytes was also measured along with the viability post-thaw to confirm cell identity. Altogether, we have demonstrated that recombinant human serum albumin can serve as a cryoprotectant in expanded T Lymphocytes to replace plasma-derived albumin and thwart the problems associated with raw material inconsistency in cellular therapies. The need for a stable final cell product to be delivered to the patient is essential to ensure product efficacy and safety with cellular therapies. It is well known that human serum albumin has critical cryoprotective activity for cell-based products in final formulations that are to be administered to the patient. However, use of plasma-derived albumin can introduce inconsistency through raw material variability which can negatively impact the final cell product efficacy and reproducibility. Recombinant human serum albumin offers an attractive solution to circumvent these challenges that arise from raw material heterogeneity in manufacturing and distribution of cell-based products. Here, we have incorporated a novel recombinant human serum albumin into an optimized chemically defined and blood-free cryopreservation media to enhance the stability of previously expanded T Lymphocytes. Cell viability was measured at multiple time points post-thaw to ensure prolonged stability of the expanded cell product. Cells cryopreserved in the chemically defined media maintained viability over time that was comparable to current industry standard final formulations. Phenotype of the cryopreserved T Lymphocytes was also measured along with the viability post-thaw to confirm cell identity. Altogether, we have demonstrated that recombinant human serum albumin can serve as a cryoprotectant in expanded T Lymphocytes to replace plasma-derived albumin and thwart the problems associated with raw material inconsistency in cellular therapies.

Abstract CT216: Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system

Abstract Scientific background/rationale: The prognosis of recurrent/refractory medulloblastoma (MB), ependymoma (EP) and atypical teratoid/rhabdoid neoplasms (AT/RT) which constitutes the majority of malignant posterior fossa tumors (MPFT) in children, remains dismal with no defined effective therapy. Current treatment strategies have significant toxicities, thus profiling newer trials with clinical efficacy and minimal side-effects are imperative. Advantages of direct infusion of Natural Killer (NK) cells into the ventricles could enhance therapeutic efficacy by maximally concentrating them in the brain in close proximity to the tumor area, bypassing the blood brain barrier and alleviating toxicity often seen with systemic chemotherapy. We have shown efficacy of activated and propagated NK cells to lyse and kill the MPFT in vitro and tumor explants in mice models. We demonstrated in a pilot study for the first time in humans, the feasibility of infusions of biologic agents into the fourth ventricle. Infusing biologic agents into the lateral ventricles are well established in neurooncology. The current ongoing study is the first-in-human, loco-regional infusion of NK cells directly into the brain evaluating safety and efficacy in the context of a phase I trial with an intent to avoid the significant side-effects of systemic therapy. This novel trial, will hopefully be able to provide therapeutic efficacy and a meaningful increase in the survival of these children. Correlative studies: Cerebrospinal fluid (CSF) withdrawn during infusions of NK cells will be evaluated for the immunophenotype, function, persistence of NK cells and influx or presence of any other immune cells. Methods: NK cells are directly infused into the lateral or fourth ventricles, after placement of a catheter at that location. Recurrent or refractory MB, AT/RT and EP involving the brain and/or spine at original diagnosis or relapse with histological verification are eligible. Three to six evaluable patients, are entered at a dose level in this study for determination of maximum tolerated dose (MTD). A minimum of 9 patients and a maximum of 24 patients are planned to be enrolled. After autologous expanded NK cell product have been manufactured as per good manufacturing guidelines (GMP) and released, patients receive 3 cycles of NK cell infusions over 12 weeks. Each cycle consists of 1 infusion per week for 3 weeks followed by a rest week (week 4). Dosing is based on recipient body surface area (BSA) and 3 dose levels will be evaluated. Currently the trial is at the final cohort (third dose level), with no dose limiting toxicity (DLT) noted with the previous dose levels. 80% of the initial minimal accrual have been met. Also as a secondary aim we will evaluate the anti-tumor activity of the NK cell infusions within the confines of a phase I study. Trial (NCT02271711-recruiting) Citation Format: Soumen Khatua, Dean Lee, Laurence Cooper, Judy Moyes, David Sandberg, Zsila Sadighi, Heather Meador, Leena Ketonen, Michael E. Rytting, Jason M. Johnson, Dristhi Raagonanan, Diane D. Liu, Vidya Gopalakrishnan, Wafik Zaky. Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system [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 CT216.

Abstract 1426: Expansion of CD8+PD-1+ T cells restored TCR repertoire as a prognostic marker to adoptive T cell immunotherapy of advanced pancreatic cancer

Abstract Purpose: We have previously reported the clinical benefits of autologous adoptive T cell immunotherapy in advanced pancreatic cancer (APC). We therefore aimed to further identify the core functional population of CD8+ T cells which exerted on the aberrant TCR repertoire and subsequently contribute to favorable prognosis after adoptive T cell therapy(ACT). Experimental Design: PBMC from APC patients (n=25) treated on a protocol of S-1 plus adoptive T cell immunotherapy were expanded ex vivo with a cytokine cocktail. The different fractions of CD8+PD-1+, CD8+LAG-3+, CD8+TIM-3+ and CD8+4-1BB+ T cells from time dependent segment were quantitated and expression of co-inhibitory molecules and biological functions on tumor killing and comparative analysis TCR repertoire from each patient were measured. The multivariate Cox hazards regression analysis was performed to identify the key T cell subset that affected the TCR repertoire recovery and subsequently associated with clinical outcome. Results: The CD3+, CD3+CD4+, and CD3+CD8+ T cells within the expanded cell product reached peak levels by day 15. CD8+ T cells exhibited enhanced expression of PD-1, LAG-3, and TIM-3, but not 4-1BB, after ex vivo expansion. Survival analysis showed that patients with a ratio of post/pre CD8+PD-1+ T cells > 2 had significantly favorable progressive-free survival (PFS) (median PFS time 180 days vs 85 days, P=0.002) and overall survival (OS) (median OS time 238 days versus 142 days, P=0.024). The sorted CD8+PD-1+ T cells displayed enhanced anti-tumor activity, and increased IFN-γ secretion after co-culture with autologous tumor cell lines. TCR repertoire diversity simultaneously rose during the ex vivo expansion and was associated with an increase in CD8+ T cells and decline in CD8+/CD28- T cells. Moreover, an increased TCR diversity was associated with significantly favorable PFS (median PFS time 166 days vs 79 days, P=0.043) and OS (median OS time 216 days versus 112 days, P=0.031) and was associated with the extent of expansion of the CD8+PD-1+ T cells (r2=0.464, P=0.001). Cox proportional hazards analysis showed that post/pre CD8+PD-1+ T cells after expansion ex vivo was an independent prognostic marker associated with both OS(P=0.009) and PFS(P=0.012). Conclusion: CD8+PD-1+ T cells, after ex vivo expansion over 15 days, are tumor-reactive. The extent of CD8+PD-1+ T cell expansion could restore TCR repertoire and was an independent prognostic factor associated with clinical outcomes in APC patients treated with ACT. Citation Format: Guoliang Qiao. Expansion of CD8+PD-1+ T cells restored TCR repertoire as a prognostic marker to adoptive T cell immunotherapy of advanced pancreatic 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 1426.

Treatment of osteonecrosis of the femoral head by core decompression and implantation of fully functional ex vivo-expanded bone marrow-derived mesenchymal stem cells: a proof-of-concept study.

BackgroundBased on several attributes involved in bone formation, bone marrow-resident mesenchymal stem cells (MSCs) have been employed in the treatment of patients suffering from femoral head osteonecrosis. Due to the low content of MSCs in the bone marrow, ex vivo expansion procedures are utilized to increase the cell number. Customarily, before administration of the resulting expanded cell product MSCs to the patient, its cellular identity is usually evaluated according to a set of “minimal phenotypic” markers, which are not modified by ex vivo processing. However, MSC functional (“reparative”) markers, which are severely impaired along the ex vivo expansion routine, are usually not assessed.Patients and methodsIn this proof-of-concept study, a cohort of five avascular osteonecrosis patients received an instillation of ex vivo-expanded autologous MSCs, manufactured under controlled conditions, with an aim to protect their functional (“reparative”) capacity.Results and conclusionOutcomes of this study confirmed the safety and effectiveness of the MSC-based therapy used. After a follow-up period (19–54 months), in all patients, the hip function was significantly improved and pain intensity markedly reduced. As a corollary, no patient required hip arthroplasty.

Immobilisation of Delta-like 1 ligand for the scalable and controlled manufacture of hematopoietic progenitor cells in a stirred bioreactor

BackgroundUmbilical cord blood provides a source of hematopoietic stem cells for transplantation with immunological and availability advantages over conventional bone marrow sources. Limited cell numbers and slower engraftment from umbilical cord blood units has led to the clinical development of immobilised Notch ligand Delta-Like 1 to promote ex vivo expansion of a rapidly engrafting cell population. However, current immobilisation methods are not simple to scale in a controlled manner.ResultsDelta-Like 1 was immobilised onto streptavidin coated magnetic particles via a heterobifunctionalised polyethylene glycol linker molecule to provide an easily manipulated format of surface protein presentation. CD34+ enriched cord blood cells were treated with Delta-Like 1 immobilised particles, and immunophenotypic markers measured to monitor population distributions using cluster identification, characterization, and regression software. The amenability of the approach to scalability was evaluated in a micro-scale stirred tank bioreactor. Surface concentration of Delta-Like 1 was well controlled used differing stoichiometric reagent ratios. Protein immobilisation was a cost effective process and particles were efficiently removed from the final cell product. Immobilised Delta-Like 1 is functional and stimulates qualitatively similar CD34hi, CD38lo, CD90lo, CD133hi, CD135hi progenitor expansion in both static culture and scalable stirred culture platforms.ConclusionsImmobilised Delta-Like 1 in this form has the potential to improve the manufacturing efficiency and control of final ex vivo expanded cell product through compatibility with highly controlled and characterised suspension culture systems.

Abstract CT033: Phase I study of intraventricular infusions of autologous exvivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system

Abstract Scientific background/rationale: The prognosis of recurrent/refractory medulloblastoma (MB), ependymoma (EP) and atypical teratoid/rhabdoid neoplasms (AT/RT), which constitutes the majority of malignant posterior fossa tumors (MPFT) in children, remain dismal with no defined effective therapy. Current treatment strategies have significant toxicities and thus profiling newer trials with clinical efficacy and minimal side-effects are imperative. Advantages of direct infusion of natural killer (NK) cells into the ventricles could enhance therapeutic efficacy by maximally concentrating them in the central nervous system (CNS) near the tumor area, bypassing the blood brain barrier and alleviating toxicity as seen with systemic chemotherapy. We have shown the efficacy of activated and propagated NK cells to lyse and kill the MPFT in vitro and tumor explants in mice models. Also, in a first-time human in vivo pilot study, we demonstrated the feasibility of infusions of biologic agents into the fourth ventricle. Infusing biologic agents through catheters placed in the lateral ventricles are well established in neurooncology. The current ongoing study is the first-in-human, loco-regional infusion of NK cells directly into the brain evaluating safety with intent to avoid the significant side-effects of systemic therapy. This novel trial will hopefully be able to provide therapeutic efficacy and a significant increase in the survival of these children. The current format of this trial incorporates changes including different dose, schedule, routes of intraventricular infusions of NK cells from the previously presented version of the trial, and thus submitted. Correlative studies: Cerebrospinal fluid (CSF) drawn during infusions of NK cells will be evaluated for the immunophenotype, function, persistence of NK cells and influx or presence of any other immune cells and correlating with tumor response. Methods: This trial (NCT02271711-recruiting) will proceed with infusions of NK cells directly into the lateral or fourth ventricles, after placement of a catheter at that location. Recurrent or refractory MB, AT/RT and EP involving the brain and/or spine at original diagnosis or relapse with histological verification will be eligible. Three to six evaluable patients may be entered at a dose level in this study for determination of maximum tolerated dose (MTD). A minimum of nine patients and a maximum of twenty-four patients will be enrolled. After autologous expanded NK cell product have been manufactured as per good manufacturing practice (GMP) guidelines and released, patients will receive three cycles of NK cell infusions over 12 weeks. Each cycle consists of one infusion per week for three weeks followed by a rest week (week 4). Dosing is based on recipient body surface area (BSA), and three dose levels will be evaluated. Currently, the trial is in the second cohort, with no dose limiting toxicity (DLT) noted with the previous dose level. 35% of the minimal accrual has been met. Also as a secondary aim, we will evaluate the antitumor activity of the NK cell infusions within the confines of a phase I study Citation Format: Soumen Khatua, Vidya Gopalakrishnan, Laurence Cooper, Dean Lee, David I. Sandberg, Michael Rytting, Jason J. Johnson, Leena Ketonen, Diane Liu, Judy Moyes, Wafik Zaky. Phase I study of intraventricular infusions of autologous exvivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT033. doi:10.1158/1538-7445.AM2017-CT033

Demonstration of Rapid Functional Myeloid Cell Recovery after Infusion of a Universal Donor Ex Vivo Expanded Progenitor Cell Therapy As a Bridging Graft Source

Cord blood transplant (CBT) recipients are known to be at risk for delayed engraftment, resulting in an increased risk of morbidity and mortality post transplant. To overcome delayed engraftment, several groups have developed methods to expand ex vivo cord blood stem/progenitor cells (HSPC) which are under clinical evaluation. The majority of these expansion methods require identification of a patient specific cord blood donor as the source material for expansion, resulting in delays in the time to transplant and inherently carry a risk of product failure. In contrast, we have developed an off-the-shelf, universal donor ex vivo expanded cord blood (CB) derived HSPC product intended for use as a transient graft source which has been demonstrated to significantly reduce the incidence of documented bacterial infections in both transplant and non-transplant settings.1,2 Donor chimerism studies conducted weekly in the first month post transplant confirm that the initial early (days 0-14) myelomonocytic engraftment is derived largely from our universal donor graft. Herein, we now demonstrate that the these rapidly engrafting myelomonocytic cells generated from the universal donor graft source are mature and functionally intact human myeloid cells that can fight infectious organisms.CBT recipients enrolled on a phase II myeloablative CBT trial were included in these ancillary studies in which we evaluated the functional capacity of newly generated myeloid cells in peripheral blood. A flow cytometry-based assay which allowed quantitation of both phagocytosis and O2-dependent killing (oxidative burst) in myeloid cells was used. Strikingly, both monocytes (CD14+) and granulocytes (CD15+) in patients’ blood displayed similar frequencies of phagocytosis and O2-dependent killing of Staphlococcus aureus at day 7 (90.3%±2.2% phagocytosis and 88.9±5.2% O2-dependent killing n=2) when more than 95% of myeloid cells were from the expanded cell product compared to day 14 (69±13.2% phagocytosis and 94±2% O2-dependent killing, n=2) when more than 99% of cells were from a non-manipulated CB unit as a result of immunologic rejection by the T cell replete CB unit. These findings provide strong evidence that de novo generated myeloid cells from expanded HSPCs are as functionally competent as myeloid cells de novo generated from non-expanded CB.To better study the functionional properties of myeloid cells derived in vivo from rapidly repopulating expanded CB HSPCs, we transplanted either 20,000 non-expanded (NE-HSPC) CD34+ CB cells or their expanded progeny (E-HSPC) into sub-lethally irradiated NOD-scid IL2rγnull (NSG) mice. At day 7 after transplantation mice transplanted with E-HSPC showed 40-fold higher human engraftment in the bone marrow than mice transplanted with NE-HSPC (28.3 ± 1% vs 0.7±0.1%, n=3, p<0.001). Remarkably, the monocytes and granulocytes from their bone marrow showed a similar phagocytic potential to that of the monocytes and granulocytes of mice receiving NE-HSPC (60.4±3.2% vs 69.6±3.2%, n=3, p=0.06). Moreover, the frequency of phagocytosis in the myeloid cells isolated from the lungs of mice receiving E-HSPC was 7-fold higher than in the lungs of mice receiving NE-HSPC. It has been well documented that E-HSPC when infused alone, also contribute to long term engraftment in NSG mice, and therefore at 22 weeks after transplantation, the frequency of phagocytosis in monocytes and granulocytes isolated from the bone marrow of mice receiving E-HSPC remained similar to that in the bone marrow of mice receiving NE-HSPC for Staphlococcus aureus (55.1 ±1.9% vs 43.8%±7%, n=5, p=0.15), Escherichia coli (50.8±2% vs 49 ±8.3%, n=5, p=0.83) and Zymosan (43.7%±3 vs 49.9%±9.2%, n=5, p=0.54) indicating the continued generation of functional myeloid cells from long term repopulating cells.We demonstrate for the first time that ex vivo expanded CB HSPCs rapidly give rise to functional myelomonocytic cells in vivo in patients and immunodeficient mice. This study validates that our universal donor off-the-shelf, expanded CB HSPC cell product is a valuable resource for patients undergoing myeloablative CBT, and further warrants its widespread use in a non-transplant setting as a supportive "myeloid bridge" to mitigate treatment-related morbidity and mortality.1. Delaney C. et al. Lancet Haematol. 2016 Jul;3(7):e330-92. Summers C. et al. Blood 2014 124:3860 DisclosuresDelaney:Nohla Therapeutics: Employment.

Clinical-scale expansion of CD34+ cord blood cells amplifies committed progenitors and rapid scid repopulation cells

Umbilical cord blood (UCB) transplantation is associated with long periods of aplastic anaemia. This undesirable situation is due to the low cell dose available per unit of UCB and the immaturity of its progenitors. To overcome this, we present a cell culture strategy aimed at the expansion of the CD34+ population and the generation of granulocyte lineage-committed progenitors. Two culture products were produced after either 6 or 14days of in vitro expansion, and their characteristics compared to non-expanded UCB CD34+ controls in terms of phenotype, colony-forming activity and multilineage repopulation potential in NOD-scid IL2Rγnull mice. Both expanded cell products maintained rapid SCID repopulation activity similar to the non-expanded control, but 14-day cultured cells showed impaired long term SCID repopulation activity. The process was successfully scaled up to clinically relevant doses of 89×106 CD34+ cells committed to the granulocytic lineage and 3.9×109 neutrophil precursors in different maturation stages. Cell yields and biological properties presented by the cell product obtained after 14days in culture were superior and therefore this is proposed as the preferred production setup in a new type of dual transplant strategy to reduce aplastic periods, producing a transient repopulation before the definitive engraftment of the non-cultured UCB unit. Importantly, human telomerase reverse transcriptase activity was undetectable, c-myc expression levels were low and no genetic abnormalities were found, as determined by G-banding karyotype, further confirming the safety of the expanded product.

Notch-Expanded Murine Hematopoietic Stem and Progenitor Cells Mitigate Death from Lethal Radiation and Convey Immune Tolerance in Mismatched Recipients.

The hematopoietic syndrome of acute radiation syndrome (h‐ARS) is characterized by severe bone marrow aplasia, resulting in a significant risk for bleeding, infections, and death. To date, clinical management of h‐ARS is limited to supportive care dictated by the level of radiation exposure, with a high incidence of mortality in those exposed to high radiation doses. The ideal therapeutic agent would be an immediately available, easily distributable single‐agent therapy capable of rapid in vivo hematopoietic reconstitution until recovery of autologous hematopoiesis occurs. Using a murine model of h‐ARS, we herein demonstrate that infusion of ex vivo expanded murine hematopoietic stem and progenitor cells (HSPCs) into major histocompatibility complex mismatched recipient mice exposed to a lethal dose of ionizing radiation (IR) led to rapid myeloid recovery and improved survival. Survival benefit was significant in a dose‐dependent manner even when infusion of the expanded cell therapy was delayed 3 days after lethal IR exposure. Most surviving mice (80%) demonstrated long‐term in vivo persistence of donor T cells at low levels, and none had evidence of graft versus host disease. Furthermore, survival of donor‐derived skin grafts was significantly prolonged in recipients rescued from h‐ARS by infusion of the mismatched expanded cell product. These findings provide evidence that ex vivo expanded mismatched HSPCs can provide rapid, high‐level hematopoietic reconstitution, mitigate IR‐induced mortality, and convey donor‐specific immune tolerance in a murine h‐ARS model. Stem Cells Translational Medicine 2017;6:566–575

Infusion of a Non HLA-Matched Off-the-Shelf Ex Vivo Expanded Cord Blood Progenitor Cell Product Following Myeloablative Cord Blood Transplantation Is Safe, Decreases the Time to Hematopoietic Recovery, and Results in Excellent Overall Survival

Background: Myeloablative cord blood transplant (CBT) recipients experience delayed hematopoietic recovery and an increased risk of transplant related mortality (TRM). With the primary goal of generating increased numbers of hematopoietic stem and progenitor cells (HSPC) to supplement a conventional cord blood (CB) graft and thereby enhance engraftment and reduce early TRM, we have developed methods to ex vivo expand CB-derived HSPC in the presence of Notch ligand for 14 days. The product is then harvested and cryopreserved for future clinical use. We have previously reported that infusion of non-HLA matched, off-the-shelf ex vivo expanded CB progenitor cells in patients undergoing myeloablative CBT is safe and associated with faster neutrophil (ANC) recovery. We now present updated outcomes data with a median follow-up of 3.2 years (range 1.9-3.7).Methods: Between September 2010 and August 2012, 15 patients with hematologic malignancies were enrolled in this single center Phase I trial to assess the safety of infusing a non-HLA matched expanded CB HSPC product to augment conventional CBT. Conditioning consisted of Fludarabine (75mg/m2), Cytoxan (120mg/kg) and TBI (13.2 Gy). On the day of transplant, 1 or 2 unmanipulated CB units were infused first followed 4 hours later by infusion of the expanded CB product. Donor(s)/host chimerism studies were performed weekly from day 7 to 28, then at days 80, 180 and 1 year on peripheral blood flow sorted into myeloid and lymphoid fractions. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. ANC and platelet engraftment were calculated using cumulative incidence rates to accommodate competing risks. Disease-free survival (DFS) was analyzed using Kaplan-Meier estimates.Results: The median age was 21 years (range, 5-45) and weight 59 kg (range, 23-89). Other patient characteristics and median infused cell doses are shown in Table 1. Four patients (26%) received only 1 unmanipulated CB unit along with the expanded graft. All patients engrafted at a median of 19 days (range, 9-31 days), significantly faster than the 25 days (range, 14-45) observed in 41 recipients of two unmanipulated units otherwise treated identically (p=0.01) (Figure 1A).Similar to our initial expansion trial using partially HLA-matched expanded CB cells, early (day 7) myelomonocytic (CD33 and CD14) recovery was almost entirely (98–100%) due to cells arising from the expansion product. Cells derived from the expansion product were no longer detected at day 14 in all but 2 patients. The median time to platelet recovery (>20 x 10^9/L) was 35 days (range 21–45).The cumulative incidence of platelet recovery by day 100 was significantly higher than that observed in our concurrent control group 92% (CI 95%: 59-99) vs. 69% (95%: 51-81), respectively (p=0.005) (Figure 1B). The probability of 3-year DFS was 86% (95% CI, 57-97). No TRM was observed throughout the study period (Figure 1C), although 2 patients relapsed at days 53 and 219 posttransplant and subsequently died after further therapy. All patients presented with grade II acute GVHD at a median time of 32 days (14-86), with no grade III-IV aGVHD observed (Figure 1D). The skin was the most commonly affected organ (n=12). Eight (53%) patients were treated for pre-engraftment syndrome at a median time of 6 days (range 4-9) and five (33%) patients had GVHD after day 100. At 2 years only 2 patients are still on immunosuppressive therapy for mild chronic GVHD.Conclusions: The results of this Phase I safety study demonstrated that infusion of an off-the-shelf non-HLA matched expanded CB HSPC product to augment a CB graft in the myeloablative setting was safe and led to more rapid neutrophil and platelet recovery compared to our concurrent control group. Importantly, no TRM was observed among the 15 patients on this study, with only 2 deaths due to relapsed disease, leading to an excellent OS of 86% at a median follow-up of 3.2 years. Quite interesting is the observation of no grade 3-4 acute GVHD, even with infusion of a non-HLA matched expanded cell product that may induce increased alloreactivity of the unmanipulated CB unit(s). However, in this preliminary pilot study, it appeared that infusion of the non HLA-matched product was associated with less severe acute GVHD. Based on these very promising results, a prospective multicenter randomized trial utilizing this product is underway. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Immunological characterization of multipotent mesenchymal stromal cells. the international society for cellular therapy (ISCT) working proposal

The large number of experimental approaches, culture conditions, qualitative and quantitative methods, and in vitro and in vivo models employed so far to assess immune regulatory properties of multipotent mesenchymal stromal cells (MSC) has led to an excess of literature data that sometimes are poorly comparable, redundant, and even contradictory. Thus, quite paradoxically, the risk is that pre-clinical literature data may become eventually weak and scarcely useful for supporting experimentally specific MSC-based clinical trials. However, some data in this field appear more solid and reproducible and may be generally accepted to suggest reproducible immunological assays to quantify the differences in immune modulatory properties of MSCs produced according to Good Manufacturing Practice (GMP). The MSC Committee of the International Society of Cell Therapy (ISCT) released a statement paper in 2006 that established the minimal criteria characterizing human MSC, without focusing particularly on their immunological properties. To consolidate the scientific research in this field, the MSC Committee of the ISCT is publishing a working proposal paper aimed at stimulating the general discussion about the need of shared guidelines for the immunological characterization of MSCs for clinical use: 1. A standard immune plasticity assay should be implemented by using IFN-γ ± TNF-α 2. Functional analysis of an expanded cell product may provide mechanistic insights on clinical response amongst patients 3. The use of purified responders would be widely practicable 4. Interrogating the IDO response as part of an in vitro licensing assay 5. Conclusions based on xenorecipient animal models should be drawn with caution 6. The prospective hypothesis-driven analysis of lymphocyte populations in patients groups treated with MSC should be encouraged 7. Clinical analysis should also include the monitoring of whether injected MSCs are the target of an immune response.

Dose Dependent Enhancement Of Neutrophil Recovery By Infusion Of Notch Ligand Ex Vivo Expanded Cord Blood Progenitors: Results Of a Multi-Center Phase I Trial

IntroductionThere is a strong clinical need to overcome the increased early non relapse mortality (NRM) associated with delayed neutrophil recovery following cord blood transplant (CBT). Therefore we established a methodology using Notch ligand (Delta1) as a strategy for increasing the absolute number of marrow repopulating CB hematopoietic stem/progenitor cells (HSPC). We previously reported preliminary results of the first 10 patients in 2010 demonstrating the ability of Notch-expanded CB HSPC to provide rapid myeloid recovery post-CBT.1 Herein we present the updated results on 23 patients accrued to this trial aimed at assessment of efficacy as well as the feasibility of overnight shipment of the expanded cell product to three outside institutions. MethodsBetween July 2006 and March 2013, 23 patients with hematologic malignancies were enrolled in this prospective multi-center Phase I trial coordinated by the Fred Hutchinson Cancer Research Center in which one CB unit was ex vivo expanded prior to infusion. Conditioning consisted of Fludarabine (75mg/m2), Cyclophosphamide (120mg/kg) and TBI (13.2 Gy) over 8 days. On day 0, the unmanipulated CB unit was infused first followed 4 hours later by infusion of the freshly harvested expanded CB cells. Graft versus host disease (GVHD) prophylaxis consisted of cyclosporine and MMF beginning on day -3. All CB grafts were 4-6/6 HLA-matched (A/B antigen level, DRB1 allele level) to the recipient. Engraftment, NRM, relapse and GVHD were calculated using cumulative incidence rates to accommodate competing risks. Overall survival was analyzed using Kaplan-Meier estimates. ResultsPatient diagnosis was AML (n=16), ALL (n=5) and biphenotypic leukemia (n=2). Nine patients (39%) were ≥CR2 and 5 were MRD+ at the time of transplant. Median age was 28 years (range, 4-43) and weight 70 kg (range, 16-91) with a median follow-up of 614 days (range, 271-2443). 22 patients received the expanded graft with one product not meeting release criteria. The cell doses infused were significantly higher in the expanded CB graft: 2.7 (1.5-6.3) vs 6.9 (0.4-27.6) x107 TNC/kg, p<0.0008; 0.15 (0.02-0.57) vs 7.7 (0.62-49.5) x106 CD34/kg, p<0.0001. HLA-matching and ABO incompatibility of the expanded and unmanipulated products were similar. The incidence of neutrophil recovery was 95% (95% CI, 71-100) at a median of 13 days (range, 6-41 days) among the 22 patients receiving expanded CB cells which is significantly faster than that observed in 40 recipients of two unmanipulated units otherwise treated identically at a median time of 25 days (range, 14 to 45; p<0.0001). The incidence of platelet recovery (>20 x 10^9/L) was 77% (CI 95%: 53- 89) by day 100 at a median of 38 days (range, 19 – 134). There was one case of primary graft failure. Importantly, rate of neutrophil recovery correlated with CD34+ cell dose/kg with 8 out of 11 patients receiving greater than 8x106 CD34+cells/kg achieved an ANC ≥ 500/µl within 10 days. 21 patients were evaluable for in vivo persistence of the expanded cells. Ten (48%) demonstrated in vivo persistence beyond one month post infusion. The expanded cell graft was persistent at day 180 in 7 patients, and in those that survived to one year, dominance of the expanded cell graft persisted in one patient. The incidences of grade II-IV and III-IV acute GVHD was 77% (95% CI, 53-89) and 18% (95% CI, 5-36%), respectively; mild chronic GVHD was observed in 4 patients and severe chronic GVHD in one. Probability of OS was 62% (95% CI, 37-79%) at 4 years. Notably, the cumulative incidence of NRM at day 100 was 8% (95% CI, 14-24%) and at 4 years was 32% (95% CI, 8-40%). Nine patients died at a median time of 216 days (range, 31-1578 days) with respiratory failure/infection the most common cause (n=6). There were two relapses at day 156 and 365 post-transplant, with one death due to relapse. Secondary malignancy and primary graft failure were the other 2 causes of death. ConclusionsInfusion of Notch-expanded CB progenitors is safe and effective, significantly reducing the time to neutrophil recovery and risks of NRM during the first 100 days. An advantage for infusion of higher numbers of CD34+ cells/kg further demonstrates the need to develop methods that reproducibly provide even greater expansion of repopulating cells than currently achieved to improve efficacy and potentially cost effectiveness.1. Delaney C, et al, Nat Med. 2010 Feb;16(2):232-6. Disclosures:Delaney:Novartis: DSMB, DSMB Other; Biolife: Membership on an entity's Board of Directors or advisory committees; medac: Research Funding. Wagner:Novartis: Research Funding; cord use: Membership on an entity's Board of Directors or advisory committees.

Immunological Characterization Of Multipotent Mesenchymal Stromal Cells. The International Society For Cellular Therapy (ISCT) Working Proposal

The large number of experimental approaches, culture conditions, qualitative and quantitative methods, and in vitro and in vivo models employed so far to assess immune regulatory properties of multipotent mesenchymal stromal cells (MSC) has led to an excess of literature data that sometimes are poorly comparable, redundant, and even contradictory. Thus, quite paradoxically, the risk is that pre-clinical literature data may become eventually weak and scarcely useful, in both researchers' and Regulatory Authorities' opinion, for supporting experimentally specific MSC-based clinical trials aimed at treating autoimmune and inflammatory diseases. However, some data in this field appear more solid and reproducible and may be generally accepted to suggest reproducible immunological assays to quantify the differences in immune modulatory properties of MSCs produced according to Good Manufacturing Practice (GMP).The MSC Committee of the International Society of Cell Therapy (ISCT) released a statement paper in 2006 that established the minimal criteria characterizing human MSC, without focusing particularly on their immunological properties. In the 7 years following the publication of this statement paper, more than 10,000 manuscripts on MSC, and many of them deal with immune regulation. To consolidate the scientific research in this field, the MSC Committee of the ISCT is publishing a working proposal paper aimed at stimulating the general discussion about the need of shared guidelines for the immunological characterization of MSCs for clinical use:1. A standard immune plasticity assay should be implemented by using IFN-γ + TNF-α as model in vitro priming agent2. Functional analysis of an expanded cell product may provide mechanistic insights on intra- and inter- study variance in clinical response amongst patients3. The use of purified responders would be widely practicable and should provide more generalizable guidance on relative functional potency of MSC and as a companion to clinical trials4. Interrogating the IDO response as part of an in vitro licensing assay should be considered central5. Conclusions based on xenorecipient animal models on how to conduct clinical trials should be drawn with caution6. The prospective hypothesis-driven analysis of lymphocyte populations in patients groups treated with MSC should be encouraged7. Clinical analysis should also include the monitoring of whether injected MSCs are the target of an immune response. Disclosures:No relevant conflicts of interest to declare.

Su1266 In Vitro Generated Regulatory T Cells From Blood Suppress Mucosal Effector T Cells in Crohn's Disease Patients

Background: Thymically derived CD4+CD25hiCD127loFOXP3+ regulatory T cells (Tregs) maintain immunological tolerance and prevent inappropriate activation of multiple immune cell types. Quantitative and qualitative Treg defects are seen in inflamed Crohn's (CD) mucosa, suggesting that insufficient Treg-mediated suppressor function contributes to chronic mucosal inflammation in CD. Consequently, cell-based therapy with autologous Tregs to restore mucosal Treg numbers and function is conceptually attractive. The recent use of in vitro expanded umbilical cord blood (UCB) Tregs to prevent GvHD following UCB transplantation confirmed the safety of this approach in humans. The optimal method of generating Tregs in vitro from CD patients to produce a homogenous, phenotypically stable yet functionally suppressive population on expansion is unknown, as is the effect of these cells on target organs: mesenteric lymph node (MLN) and lamina propria (LP) immune cells in inflamed CD gut. Aim: To define the optimal method of GMP-compatible Treg isolation and expansion from CD patients' blood with reference to homogeneity, phenotypic stability and suppressive ability of the cell product. Results: Tregs were expanded for 2028 days in X-VIVO15 with 5% human AB serum, IL-2 and Rapamycin. Tregs initially isolated using GMP-compatible MACS-based enrichment had unacceptably high CD8+ contamination (.20%) and high expression of IL-17 and IFNγ (n=5 independent experiments). CD4+CD25+CD127lo Tregs were then sorted from CD blood on the basis of CD45RA expression and expanded a median of 127-fold (IQR 58-531) to yield a .97% CD4+CD25hiCD127loFOXP3+ population (n=10). Expanded CD45RA+ Tregs expressed functionally relevant markers including CTLA-4, ICOS, CD27 and CD62L ( .97% each) and TGF-β but not IL-10. CD45RA+ Tregs expressed ,1% IL-17 by FACS and had greater phenotypic stability than expanded CD45RATregs, with less spontaneous (p,0.05) and induced (p,0.01) IL-17 production by ELISA. CD45RA+ Tregs highly suppressed autologous CD4+CD25proliferation at 96h (median=97.3% [96.6%-99.1%] at 1:1 ratio); and were more effective than CD45RATregs at suppressing CD4+CD25activation (p=0.003), assessed by CD154 expression at 7h. Importantly, using CD3+ T responders from inflamed Crohn's resections, CD45RA+ Tregs suppressed MLN CD3+ proliferation (88.4% [53.8-93.2%]), MLN CD154 expression (82.9% [60.1-87.6%]) and LP CD3+ CD154 expression (64.3% [64.377 . 9%] ) i n a do s e d ep enden t manne r . Conc lu s i ons : FACS so r t i n g o f a CD4+CD25hiCD127loCD45RA+ precursor population is required to produce a homogenous, phenotypically stable in vitro expanded cell product from CD patients. For the first time, we show that in vitro expanded Tregs suppress immune cell activation in relevant mucosal immune niches, strengthening the rationale for clinical trials of autologous in vitro expanded Tregs in CD.

Infusion of “off-the-shelf” Third Party Ex Vivo Expanded Cord Blood Progenitor Cells As Supportive Care Following Clofarabine with High Dose Cytarabine and Granulocyte Colony-Stimulating Factor Priming for the Treatment of AML,

Abstract 3640Regardless of patient age, prolonged neutropenia and infectious complications are common side effects of AML induction and salvage chemotherapy, requiring intensive supportive care and contributing to treatment failure. Thus, novel therapies that can abrogate prolonged pancytopenia and facilitate more rapid hematopoietic recovery are needed. Previously, we have demonstrated that the absolute number of repopulating cord blood (CB) hematopoietic stem/progenitor cells (HSPC) can be increased by culture with Notch ligand; recent data from a Phase I double CB transplant trial utilizing ex vivo expanded CB HSPC has suggested more rapid neutrophil recovery (Delaney et al, Nat med). We hypothesize that this expanded cell product, which is devoid of T cells, could be cryopreserved and then infused as an off-the-shelf non-HLA matched product to provide rapid but temporary donor myeloid engraftment and might also facilitate autologous hematopoietic recovery following AML therapy, thus reducing the infectious complications associated with therapy.In a cohort of 50 patients, Becker et al at the Fred Hutchinson Cancer Research Center have reported on the use of clofarabine and high dose ara-c, in combination with G-CSF (GCLAC) in a phase I/II trial for the treatment of relapsed/refractory AML (Becker et al, Br. J. Haematol, in press). GCLAC is profoundly immunosuppressive and myelosuppressive, with periods of prolonged neutropenia post-GCLAC ranging from 17 to 35 days (median time to ANC >500 is 21 days) in this cohort. Infection was the most frequent adverse event, with ≥ grade 3 bacterial and fungal infections seen in 40% of patients (including 1st and ≥ 2nd salvage patients). We now herein report the preliminary results of a phase I safety trial investigating the use of GCLAC plus infusion of off-the-shelf expanded and cryopreserved CB HSPC for patients with AML.To date, 12 adult patients with relapsed (n=8), primary refractory (n=2) or de novo high risk (n=2) AML have been enrolled and received GCLAC as their first salvage or initial induction therapy. The median age and weight was 55 years (range, 38 to 59 years) and 79.5 kg (range, 63.8 to 126) respectively. Patients received cycle 1 of GCLAC followed by infusion of off-the-shelf expanded CB HSPC 24 hours after completion of chemotherapy. Patients who achieved a complete morphologic remission and did not experience any toxicities associated with the expanded cell product were eligible to receive a 2nd cycle of GCLAC with cells if not proceeding directly to stem cell transplant. A total of 15 products have been infused in 12 patients. Five of the 12 patients were refractory to GCLAC and therefore non-evaluable for neutrophil recovery. The median time to achieve an ANC > 500 in the evaluable patients was 19 days (range 18 to 27) after cycle 1, as compared to 21 days (range 17 to 35) in the historical cohort of patients receiving GCLAC only without expanded cells. Importantly, in this phase I study, there have been no infusional toxicities from the expanded cells or adverse events attributed to the expanded cell product. Alloimmunization has not been observed as monitored by pre- and post-infusion panel reactive antibody assays to determine the presence of donor directed antibodies.Four of the 12 enrolled patients have experienced clinically significant infections (e.g., bacteremia, fungal infections) compared to 40% in the comparison cohort, and no infectious deaths have been observed. Finally, in contrast to the first cycle of chemotherapy where donor cells were detected in only one patient, 4 out of 4 patients who received a 2nd cycle of GCLAC with expanded cells were found to have transient donor contribution to myeloid recovery (by peripheral blood cell sorted DNA chimerism assay) one week after infusion of the cells ranging from 85 to 100% donor in the CD33/CD14 cell lineages. Expanded cells were also able to home to the marrow as evidenced by transient donor engraftment (range, 3 to 15%) in recipient marrows one week after expanded cell infusion. It is unclear whether this is related to increased marrow “space” or increased host immunosuppression following cycle 2. With this encouraging data, we are continuing accrual of newly-diagnosed patients. This approach may decrease complications of pancytopenia and allow delivery of more dose-intense therapy, particularly in patients with intermediate/favorable cytogenetics who are already relatively sensitive to GCLAC. Disclosures:Delaney:Sanofi-Oncology (formerly Genzyme): Clofarabine for the study supplied by Sanofi-Oncology (formerly Genzyme). Becker:Sanofi-Oncology (formerly Genzyme): Research Funding.

ASH 2010 meeting report—Top 10 clinically oriented abstracts in transfusion medicine

ASH 2010 meeting report—Top 10 clinically oriented abstracts in transfusion medicine

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

AbstractAbstract 340Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB.The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%).We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone.We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populationsHistone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells.The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4.We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures:No relevant conflicts of interest to declare.

Retention of Quiescent Hematopoietic Cells With High Proliferative Potential During Ex Vivo Stem Cell Culture

Human CD34+/Thy-1+/Lin- hematopoietic cells purified from bone marrow (BM) or mobilized peripheral blood (MPB) are highly enriched for pluripotent stem cells. Ex vivo expansion of this population is proposed as a means of providing accelerated short-term, as well as long-term, engraftment after myeloablative therapy. Here we demonstrate that primitive quiescent cells are retained in bulk expansion cultures of CD34+/Thy-1+/Lin- cells and that the cell production capacity of the expanded cell product can largely be attributed to cells exhibiting quiescent behavior during culture. CD34+/Thy-1+/Lin- cells from adult BM or MPB were labeled with the fluorescent membrane dye PKH26, followed by in vitro culture of 10(4) cells on a murine stromal layer in the presence of interleukin (IL)-3, IL-6, c-kit ligand (KL), and leukemia inhibitory factor (LIF). With each subsequent cell division, PKH26 fluorescence is reduced by roughly half, which allows tracking of the number of cell divisions. Progenitor cells present after a 2-week expansion period were sorted into CD34+/Lin-/dyebright and CD34+/Lin-/dyedim fractions and then cultured in a 4-week single-cell proliferation assay to characterize the proliferative capacity of each group. Fifty-nine percent of progenitors remaining dyebright after bulk culture (four or fewer cell divisions) were observed to proliferate in single cell culture, and produced an average of 1,780 cells per plated cell. In contrast, only 26% of dyedim (more than four divisions) progenitors were observed to proliferate and displayed a lower average proliferative capacity of 225 cells per plated cell. Similar behaviors were observed after a second consecutive cycle of bulk culture, indicating that quiescent cells with high proliferative capacity existed in culture for at least 4 weeks. Single CD34+/Lin-/dyebright progenitors purified from bulk cultures were observed to produce as many as 1,000 CD34 positive progeny during single cell culture, and these progeny included multilineage colony forming cells. These data demonstrate that among CD34 positive cells recovered after in vitro bulk culture, a higher proliferative capacity correlated with quiescent behavior. The described culture method provides quantitation of the cell producing capacity of individual cells in hematopoietic cell mixtures and may prove useful for predicting engrafting potential in products intended for cellular therapy.