Mobilized Peripheral Blood Cells Research Articles

MPN-238 Single-Cell RNA Profiling of Myelofibrosis Patients Reveals Pelabresib-Induced Decrease of Megakaryocytic Progenitors and Normalization of CD4+ T Cells in Peripheral Blood.

Abnormal differentiation of the megakaryocytic lineage and overproduction of proinflammatory cytokines, resulting in bone marrow fibrosis, anemia, symptoms, extramedullary hematopoiesis, and often hepatosplenomegaly, are key characteristics of myelofibrosis. Bromodomain and extraterminal domain (BET) proteins play a significant role in the regulation of neoplastic myeloproliferation and proinflammatory cytokine production. Pelabresib (CPI-0610) is an investigational, oral, small-molecule BET inhibitor. Characterize cellular composition and transcriptional alterations in peripheral blood (PB) obtained from myelofibrosis patients enrolled in three arms (Arm 1: pelabresib monotherapy; Arm 2: pelabresib 'add-on' to ruxolitinib in patients with suboptimal ruxolitinib response; Arm 3: pelabresib with ruxolitinib in JAKi-naïve patients) of the ongoing MANIFEST Phase 2 study (NCT02158858). We performed single-cell RNA sequencing on 234,904 CD34+ HSPCs and 135,970 CD34- mature PB cells. Baseline and on-treatment samples were obtained from a random pool of 20 patients (Arm 1: n=5; Arm 2: n=8; Arm 3: n=7). Mobilized PB cells from healthy donors (HD) were used as controls (n=11). Analysis of CD34+ HSPCs at baseline demonstrated increased numbers of megakaryocytic, neutrophilic, and erythroid progenitors in myelofibrosis patients compared with HDs and a significant reduction of myeloid and B-cell lineage progenitors. Pelabresib as monotherapy and combined with ruxolitinib led to significant reduction of megakaryocytic, neutrophilic, and erythroid progenitors compared with baseline. Analysis of CD34- cells from myelofibrosis patients identified a significantly lower proportion of CD4+ T cells and increased numbers of erythroid cells at baseline versus HD. Individual patients exhibited reduction in natural killer cells and CD16+ monocytes as well as elevated megakaryocytic lineage cells. Pelabresib as monotherapy and combined with ruxolitinib increased the proportion of CD4+ T cells, and, importantly, reduced megakaryocytic lineage cells in both treatment-naïve and ruxolitinib-relapsed/refractory patients. In myelofibrosis patients at baseline, larger spleen volume was observed in patients with lower numbers of CD4+ T cells and increased numbers of megakaryocytic and myeloid CSF3R+ cells. Single-cell profiling of a subset of myelofibrosis patients in the MANIFEST study suggests that pelabresib alone and in combination with ruxolitinib induces improvement of the myeloid-lymphoid imbalance. This potential disease-modifying effect warrants further investigation.

S192: SINGLE-CELL RNA PROFILING OF MYELOFIBROSIS PATIENTS REVEALS PELABRESIB-INDUCED DECREASE OF MEGAKARYOCYTIC PROGENITORS AND NORMALIZATION OF CD4+ T CELLS IN PERIPHERAL BLOOD

Background: Abnormal differentiation of the megakaryocytic (MK) lineage in conjunction with overproduction of proinflammatory cytokines (Ck), resulting in bone marrow fibrosis, anemia, extramedullary hematopoiesis and often hepatosplenomegaly, are the main biological and clinical characteristics of myelofibrosis (MF). Bromodomain and extraterminal domain (BET) proteins play a key role in the regulation of neoplastic myeloproliferation and proinflammatory Ck production. Pelabresib (CPI-0610) is a potent, selective BET inhibitor under investigation in MF patients (pts) as monotherapy and in combination with the Janus kinase inhibitor (JAKi) ruxolitinib (RUX) in the ongoing MANIFEST Phase 2 study (NCT02158858). Arm 1: pelabresib as monotherapy in RUX-intolerant, ineligible or refractory MF pts; Arm 2: pelabresib in combination with RUX as an ‘add-on’ in MF pts with suboptimal response to RUX; Arm 3: pelabresib combination therapy with RUX in JAKi-naïve MF pts. Pelabresib as monotherapy and in combination with RUX improved spleen volume reduction, symptoms and hemoglobin levels (Talpaz M, ASH 2020). The effects of pelabresib on hematopoietic stem/progenitor cells (HSPCs) and immune cell populations in MF pts are presented here. Aims: We aimed to characterize the cellular composition and transcriptional alterations occurring in peripheral blood (PB) obtained from MF pts enrolled in the MANIFEST trial. Methods: We performed single-cell RNA sequencing on 234,904 CD34+ HSPCs and 135,970 CD34- mature PB cells (Figure). Cells were obtained from a random pool of 20 pts (Arm 1 n=5, Arm 2 n=8, Arm 3 n=7), which included a baseline sample (BL) and samples collected during treatment (range: C3 through C12) for each pt. Mobilized PB cells from healthy donors (HD) were used as a control (n=11). Results: Analysis of CD34+ HSPCs at BL demonstrated a statistically significant increase of MK, neutrophilic and erythroid progenitors in MF pts compared with HDs and decreased numbers of myeloid and B cell lineage progenitors. Pelabresib as monotherapy and in combination with RUX led to a significant reduction of MK-, neutrophilic and erythroid progenitors as compared with BL in all pts analyzed. Analysis of CD34- cells from MF pts identified a significantly lower proportion of CD4+ T cells and increased numbers of erythroid cells at BL compared with HD. Individual pts also exhibited reduction in natural killer cells and CD16+ monocytes as well as elevated MK lineage cells. Pelabresib as monotherapy and in combination with RUX increased the proportion of CD4+ T cells, and more importantly, reduced MK lineage cells compared with BL in both treatment-naïve and RUX relapsed/refractory (r/r) pts. In MF pts at BL, a larger spleen volume was observed in pts with lower numbers of CD4+ T cells and increased numbers of MK- and myeloid CSF3R+ cells. Updated results of the cell composition analysis and transcriptional reprogramming during pelabresib treatment, as well as correlation with effects on cytokine plasma levels and clinical efficacy data, will be presented. Image:Summary/Conclusion: The single-cell profiling of a subset of MF pts enrolled in the MANIFEST study suggests that pelabresib alone and in combination with RUX induces an improvement of the myeloid-lymphoid imbalance in both JAKi-naïve and r/r MF pts. The observed effects of pelabresib on HSPCs from MF pts confirm the ex vivo activity on erythroid and MK differentiation of stem cells (Keller P, EHA 2021; Verstovsek S, ASH 2021) and implies a potential disease-modifying effect warranting further investigation.

Short treatment of peripheral blood cells product with Fas ligand using closed automated cell processing system significantly reduces immune cell reactivity of the graft in vitro and in vivo

Mobilized peripheral blood cells (MPBCs) graft and peripheral blood cells apheresis are used for bone marrow transplantation and for treatment of graft versus host disease (GvHD). We demonstrate that a short treatment of MPBCs with Fas ligand (FasL, CD95L) for 2 h using a closed automated cell processing system selectively induces apoptosis of specific donor T cells, B cells and antigen presenting cells, but, critically, not CD34+ hematopoietic stem cells and progenitors, all of which may contribute to an increased likelihood of graft survival and functionality and reduced GvHD. Treated cells secreted lower levels of interferon-gamma as compared with control, untreated, cells. Moreover, FasL treatment of immune cells increased signals, which led to their phagocytosis by activated macrophages. FasL treated immune cells also reduced the ability of activated macrophages to secrete pro-inflammatory cytokines. Most importantly, FasL ex vivo treated MPBCs prior to transplantation in NOD-SCID NSG mice prevented GvHD and improved stem cell transplantation in vivo. In conclusion, MPBCs, as well as other blood cell products, treated with FasL by automated manufacturing (AM), may be used as potential treatments for conditions where the immune system is over-responding to both self and non-self-antigens.

Restoration of aged hematopoietic cells by their young counterparts through instructive microvesicles release.

This study addresses the potential to reverse age-associated morbidity by establishing methods to restore the aged hematopoietic system. Parabiotic animal models indicated that young secretome could restore aged tissues, leading us to establish a heterochronic transwell system with aged mobilized peripheral blood (MPB), co-cultured with young MPB or umbilical cord blood (UCB) cells. Functional studies and omics approaches indicate that the miRNA cargo of microvesicles (MVs) restores the aged hematopoietic system. The in vitro findings were validated in immune deficient (NSG) mice carrying an aged hematopoietic system, improving aged hallmarks such as increased lymphoid:myeloid ratio, decreased inflammation and cellular senescence. Elevated MYC and E2F pathways, and decreased p53 were key to hematopoietic restoration. These processes require four restorative miRs that target the genes for transcription/differentiation, namely PAX and phosphatase PPMIF. These miRs when introduced in aged cells were sufficient to restore the aged hematopoietic system in NSG mice. The aged MPBs were the drivers of their own restoration, as evidenced by the changes from distinct baseline miR profiles in MPBs and UCB to comparable expressions after exposure to aged MPBs. Restorative natural killer cells eliminated dormant breast cancer cells in vivo, indicating the broad relevance of this cellular paradigm - preventing and reversing age-associated disorders such as clearance of early malignancies and enhanced responses to vaccine and infection.

Safety and Preliminary GvHD Mitigation in First in Human Clinical Trial of Apograft in Match Related Stem Cell Transplantation (SCT)

Graft versus host disease (GvHD) is a major frequent adverse event (AE) and the primary cause of morbidity and mortality following allogeneic stem cell transplantation (ASCT).Numerous attempts to reduce incidence and severity of GvHD using a variety of cell selection methods have been employed, but they all suffer from a risk/benefit trade-off where reduction of GvHD-causing cells leads to reduced engraftment and/or reduced graft vs. tumor (GvT) effect.We have previously reported development of a new method for cell selection, which take advantage of cells' differential sensitivity to CD95 receptor mediated apoptosis. This technology enables the differential selection of transplant related immune-toxicity cells (e.g., TH1, TH17, terminally differentiated B cells and antigen presenting cells {APCs}) while maintaining stem and progenitor cells. Thus, selecting the right mix of cells may enable both GvHD reduction and maintenance of the graft's effectiveness.Here we report results of our first in humans clinical trial using this functional cell selection technology (named ApoGraft). This study was an Open-Label Phase 1/2 Pilot, Staggered Four-Cohort Safety and Proof-of-Concept Study of ApoGraft in the Prevention of Acute Graft Versus Host Disease in Match related SCT (NCT02828878). The primary objective of the trial was to demonstrate safety and tolerability of ApoGraft in adult patients with hemato-oncology disorders. The key secondary objective was engraftment and prevention of acute GvHD.Eleven patients were enrolled into this study in 4 sequential cohorts of 3 patients each, except for 2 patients in cohort 4. Enrolled subjects' transplant indications included AML (n=7), ALL (n=1), MDS (n=2) and biphenotypic acute leukemia (n=1). ApoGraft was manufactured ex-vivo from donor mobilized peripheral blood cells (MPBCs) collected via a single apheresis donation that was incubated for 2 hours with FasL protein (10, 25, 50 and 100 ng/ml per cohort), and washed before transplantation to the recipient. Patients were administered with a single ApoGraft treatment. The ApoGraft product median CD34+ yield was 72%, with a median of 4.3x10^6 CD34+ cells/Kg/patient. All patients received GVHD prophylaxis with short-course methotrexate and Cyclosporin A.No AEs or serious AEs (SAEs) were recorded during stem cell infusion. Six months follow-up showed ApoGraft transplantation to be well tolerated with no related AEs or SAEs. Successful engraftment was observed in all subjects within the expected timeframe. Median time to neutrophils or platelets engraftment was 14 days. Lower incidence of grade 2-4 aGvHD was observed in Cohorts 3 and 4 (n=2) as compared to Cohorts 1 and 2 (n=5). None of the higher FasL dose Cohort 3 and Cohort 4 patients had grade 3 and 4 aGvHD compared to 3 patients in cohorts 1 and 2. Over 97% of bone marrow at 28 days post-transplant were of donor origin, indicating full donor chimerism, except for the two MDS patients; one had shown full donor chimerism, which was reduced at day 180 (55%). The other had a donor chimerism of 92% at day 28. None of the patients relapsed during the study period. Four of 6 patients in the 10 and 25 ng/ml cohorts had 7 episodes of CMV viremia, as compared with 1 episode in the 50ng/ml cohort and none in the 100ng/ml cohort. Ten patients survived and were progression-free by study day 180. One MDS patient died during the study due to study unrelated secondary graft failure.Our results show evidence that the ApoGraft product was well tolerated at all FasL doses and demonstrated preliminary signs of GvHD mitigation when the higher ex-vivo doses of FasL (50 and 100 ng/ml) were used. DisclosuresZuckerman: AbbVie: Honoraria; Orgenesis Inc.: Honoraria; Janssen: Honoraria; BioSight Ltd: Honoraria; Novartis: Honoraria; Gilead Sciences: Honoraria, Speakers Bureau; Cellect Biotechnology: Honoraria. Yehudai-Ofir: Novartis: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees. Kamar: Cellect Biotheraputics: Consultancy.

Results of a Non-Interventional Clinical Trial of Apograft — a Novel Technology for GvHD Prevention

IntroductionGvHD is a threat to the wellbeing and survival of patients following hematopoietic stem cell transplantation (HSCT). In-vivo and ex-vivo T cell depletion are the most effective methods for GvHD prevention, but attendant risks of infection, graft failure and leukemic recurrence offset the advantages of these manipulations. Less extensive T cell depletion leaves many patients at risk for the ravages of treatment-resistant GvHD. Alternative approaches to eliminate GvHD-causing cells are needed. Fas ligand (FasL), a member of the tumor necrosis factor (TNF) family, can selectively induce apoptosis of mature T cells while sparing stem and progenitor cells. We have developed a novel FasL-mediated selection process which eliminates mature T cell subsets in hematopoietic stem cell grafts and effectively prevents GvHD in pre-clinical models without impairing engraftment and immune system recovery. We report here data from ex-vivo and in-vivo studies supporting the use of FasL-mediated depletion of mature T-cells, a process we have named ApoGraft, as a novel effective method for GvHD prophylaxis in the context of allogeneic HSCT.Methods:Aliquots of mobilized peripheral blood cells (MPBC) were collected from 104 consenting healthy donors and were used ex-vivo for optimization of the ApoGraft manufacturing process. ApoGraft is manufactured under GMP guidelines, and includes two washing steps, a 2 hr incubation of the graft at 37oC with human recombinant different concentrations of FasL (MegaFasL, Adipogen), and 2 additional washing steps to remove unbound FasL. Aliquots from 25 MPBC apheresis that had been subjected to the complete ApoGraft process were analyzed ex-vivo for cell yield, viability and cellular constitution by FACS and for clonogenicity potential by colony forming unit (CFU) assays. Additionally, engraftment and GvHD prevention of ApoGraft were evaluated with 7 aliquots of MPBC apheresis in NOD-SCID IL2-Rgamma-null (NSG) mice (n=349). Here we report results from representative studies were unfractionated ApoGraft (2.5x10^6 cells) or CD34+ purified cells (1.0x10^5 cells) from ApoGraft were transplanted into irradiated (2-2.75Gy) NSG mice. The mice were monitored clinically for GVHD occurrence post transplantation; engraftment and differentiation were evaluated using FACS analysis.Results:Cellular viability was 98% (±1%) in the ApoGraft samples analyzed. Comparison of ApoGraft and Starting Material (SM) showed a mean 19.9% (±4.3%) reduction of total CD3+ T cells; early apoptosis of CD3+ cell increased by 5-fold as compared to Starting Material. Importantly, the percentage of viable CD34+ stem and progenitor cells was not affected by ApoGraft processing. Proliferation and differentiation of hematopoietic progenitors in colony forming unit (CFU) assay was not affected. In NSG mice infused with unfractionated ApoGraft or CD34+ cells purified from it, chimerism level and differentiation were similar to those of mice infused with control MPBC (figure 1A-1B). Most importantly, ApoGraft infused mice had lower GvHD clinical score and prolonged survival as compared to mice infused with control MPBC (maximal score 1.7±0.7 vs. 8.8±0.8) (Figure 1C).Conclusion:ApoGraft is an apoptosis-based selection process that eliminates mature T cells from stem cells grafts, while preserving CD34+ cells quantity, quality and functionality. In our preclinical murine model, ApoGraft reduced GvHD without impairing engraftment. Ongoing animal studies are evaluating the graft vs. leukemic capabilities of ApoGraft and the preservation of anti-viral immunity after ApoGraft processing. Additionally, ApoGraft is currently being evaluated in a Phase I/IIa study (NCT02828878) in subjects with hematological malignancies undergoing matched related allogeneic HSCT. [Display omitted] DisclosuresZuckerman:BioSight Ltd.: Consultancy. Yarkoni:Cellect Biotechnology Ltd.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Nechushtan:Cellect Biotechnology Ltd.: Employment. Rodionov:Cellect Biotechnology Ltd.: Employment. Gez:Cellect Biotechnology Ltd.: Employment. Rosenzwaig:Cellect Biotechnology Ltd.: Employment. Rodin:Cellect Biotechnology Ltd.: Employment. Pereg:Cellect Biotechnology Ltd.: Employment.

Restoration of Aged Hematopoietic Cells by Their Young Counterparts Through Instructive Microvesicle Release

This study addresses the potential to reverse age-associated morbidity by establishing methods to restore the aged hematopoietic system. Parabiotic animal models indicated that young secretome could restore aged tissues, leading us to establish a heterochronic transwell system with aged mobilized peripheral blood (MPB), co-cultured with young MPB or umbilical cord blood (UCB) cells. Functional studies and omics approaches indicate that the miRNA cargo of microvesicles (MVs) restores the aged hematopoietic system. The in vitro findings were validated in immune deficient (NSG) mice carrying an aged hematopoietic system, improving aged hallmarks such as increased lymphoid:myeloid ratio, decreased inflammation and cellular senescence. Elevated MYC and E2F pathways, and decreased p53 were key to hematopoietic restoration. These processes require four restorative miRs that target the genes for transcription/differentiation, namely PAX and phosphatase PPMIF. These miRs when introduced in aged cells were sufficient to restore the aged hematopoietic system in NSG mice. The aged MPBs were the drivers of their own restoration, as evidenced by the changes from distinct baseline miR profiles in MPBs and UCB to comparable expressions after exposure to aged MPBs. Restorative natural killer cells eliminated dormant breast cancer cells in vivo, indicating the broad relevance of this cellular paradigm - preventing and reversing age-associated disorders such as clearance of early malignancies and enhanced responses to vaccine and infection.

Multicolor Flowcytometry Analysis of Hematopoietic Stem and Progenitor Cells Subsets Among Basal and Mobilized Peripheral CD34+ Cells

BACKGROUNDIn the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation.Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODSmulticolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTSPercentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p<0.005 and p<0.01).No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs.Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p<0.0001) in cyclophosphamide treated patients, compared to patients mobilized with other chemotherapy regimens. The percentage of HSCs did not significantly differ among bone marrow, unmobilized and mobilized PB CD34+ cells. Therefore, since an average collection of mobilized PB cells contains approximately one log more CD34+ cells than a BM harvest, a similarly higher amount of HSC are infused with mobilized CD34+ cell transplantation.A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONSOur data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. DisclosuresPalumbo:Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

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

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

Hematopoietic stem cell origin of human fibroblasts: Cell culture studies of female recipients of gender-mismatched stem cell transplantation and patients with chronic myelogenous leukemia

Our series of studies using transplantation of single hematopoietic stem cells (HSCs) demonstrated that mouse fibroblasts/myofibroblasts are derived from HSCs. In order to determine the origin of human fibroblasts, we established a method for culturing fibroblasts from human peripheral blood (PB) mononuclear cells and studied fibroblasts from gender-mismatched HSC transplant recipients and patients with untreated Philadelphia chromosome-positive chronic myelogenous leukemia (CML). We cultured PB cells from three female subjects who showed near-complete hematopoietic reconstitution from transplantation of granulocyte-colony stimulating factor-mobilized male PB cells and examined the resulting fibroblasts using fluorescent in situ hybridization for Y chromosome. Because the mobilized PB cells may contain mesenchymal stem cells, we could not determine the HSC or mesenchymal stem cell origin of the fibroblasts seen in culture. To further document the HSC origin of human fibroblasts, we next examined fibroblasts from two patients with untreated CML, a known clonal disorder of HSCs. All cultured fibroblasts from female recipients of male cells showed the presence of Y chromosome, indicating the donor origin of fibroblasts. Cultured fibroblasts from the CML patients revealed the presence of BCR-ABL translocation. This demonstration provided strong evidence for the HSC origin of human fibroblasts because CML is a clonal disorder of the HSC. These studies strongly suggest that human fibroblasts are derived from HSCs. In addition, the results suggest that fibrosis seen in patients with CML may be a part of the clonal process.

Sustained Long-Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Third-Generation Lentiviral Vectors

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.

Nucleolin Regulates Gene Expression in CD34-positive Hematopoietic Cells

CD34 glycoprotein in human hematopoiesis is expressed on a subset of progenitor cells capable of self-renewal, multilineage differentiation, and hematopoietic reconstitution. Nucleolin is an abundant multifunctional phosphoprotein of growing eukaryotic cells, involved in regulation of gene transcription, chromatin remodeling, and RNA metabolism, whose transcripts are enriched in murine hematopoietic stem cells, as opposed to differentiated tissue. Here we show that, in human CD34-positive hematopoietic cells, nucleolin activates endogenous CD34 and Bcl-2 gene expression, and cell surface CD34 protein expression is thereby enhanced by nucleolin. Nucleolin-mediated activation of CD34 gene transcription results from direct sequence-specific interactions with the CD34 promoter region. Nucleolin expression prevails in CD34-positive cells mobilized into peripheral blood (PB), as opposed to CD34-negative peripheral blood mononuclear cells (PBMCs). Therefore, in intact CD34-positive mobilized PB cells, a recruitment of nucleolin to the CD34 promoter region takes place, accompanied by nucleosomal determinants of gene activity, which are absent from the CD34 promoter region in CD34-negative PBMCs. Our data show that nucleolin acts as a component of the gene regulation program of CD34-positive hematopoietic cells and provide further insights into processes by which human CD34-positive hematopoietic stem/progenitor cells are maintained.

Inherent Differences in SDF-1α-Induced CXCR4 Expression Patterns on MPB, BM and CB CD34+ Cells.

We have previously shown that in vitro migration and NOD/SCID engraftment potential of mobilized peripheral blood (MPB) CD34+ cells can be significantly increased 2–3 fold by preincubation of graft cells with 100 ng/ml of SDF-1α (CXCL-12) for 30mins at 4°C. However, bone marrow (BM) and cord blood (CB) CD34+ cells did not exhibit similar increases in migration or engraftment after SDF-1α preincubation and, in the case of CB, preincubation somewhat reduced in vitro migration. We believe that these biological differences in SDF-1α responses may begin to explain some of the reported differences in hematopoietic potential among these three sources of CD34+ cells, and may be related to rate and degree of CXCR4 receptor recycling after SDF-1α exposure. In the present work, we examined patterns of CXCR4 expression both before (part 1 studies) and after (part 2 studies) SDF-1α pretreatment, and found that, in general, MPB CD34+ cells appear to possess an inherent ability to mobilize internal CXCR4 stores with greater efficiency than BM and CB CD34+ cells, while CB appears most sensitive to SDF-1α-induced CXCR4 downmodulation. This conclusion is supported by the following results: In part 1 studies, when untreated CD34+ cells were incubated with SDF-1α (100 ng/ml, 37°C), CXCR4 expression initially decreased in all tissues, but returned to basal levels in BM and MPB cells, while re-expression on CB cells remained below baseline for the duration of the 2hr assay. In parallel cultures where SDF-1α was washed out after 60mins, CXCR4 re-expression was most rapid in MPB CD34+ cells, reaching 4.5±2.7-fold higher levels of expression after 30mins, compared to 1.8±1.1-fold for BM (p<0.05) and 3.4±2.0 for CB. After SDF-1α pretreatment (part 2 studies) and subsequent 37°C culture, CXCR4 expression declined initially in all three tissues, but was rapidly regained in MPB such that the fold increase above basal levels was 2-fold higher compared to BM or CB. CB CD34+ cells again exhibited a sustained decline of CXCR4 expression below baseline for up to 2hr. In the presence of SDF-1α, preincubated CB CD34+ cells again showed the greatest reduction in CXCR4 expression to <50% of baseline levels compared to BM and MPB, where re-expression reached basal values (p<0.05). Additionally, CXCR4 re-expression after SDF-1α wash-out was significantly increased more than 3-fold in MPB CD34+ cells compared to BM. The lower percentage of fresh MPB CD34+ cells expressing surface CXCR4 compared to BM and CB (%CXCR4+ = 9.6±3.5, 28.4±5.9 and 28.1±6.9, respectively, p<0.05, n=6–8), may contribute to the pronounced effect of SDF-1α on MPB, considering that the activity level of the recycled receptor may differ from that on fresh cells, a topic currently under study in our laboratory. Taken together, these results suggest that after SDF-1α preincubation, MPB CD34+ cells retain their ability to rapidly mobilize CXCR4 from internal stores to the cell surface, while BM and CB responses are more blunted than prior to SDF-1α pretreatment. In addition, exposure of CB CD34+ cells to SDF-1α results in prolonged down-modulation of the SDF-1α/CXCR4 axis manifested by internalization of CXCR4. These data highlight key biological differences in the SDF-1α/CXCR4 axis in CD34+ cells from MPB, BM, and CB that may begin to explain some of the observed differences in their hematopoietic potential. Furthermore, these findings may be important in the design of clinical transplantation protocols utilizing specific SDF-1α-pretreatment protocols to maximize the clinical benefit of transplanted HSC from different sources.

Engraftment kinetics of human CD34+ cells from cord blood and mobilized peripheral blood co-transplanted into NOD/SCID mice

We have reported short periods of post transplant neutropenia in human patients co-transplanted with cord blood (CB) and low numbers of haploidentical mobilized peripheral blood (MPB) CD34+ cells. To investigate the effect that the proportion of MPB to CB cells may have on engraftment kinetics, we have co-transplanted fixed numbers of human CB CD34+ cells mixed with different numbers of MPB CD34+ cells into NOD/SCID mice. We periodically quantified the proportion of human cells and the relative contribution of MPB and CB cells to the human engraftment on marrow aspirates. At the lowest MPB/CB ratios (5 : 1, 10 : 1), the contribution of CB cells predominated at all time points analyzed, and in three out of four experiments MPB cell contributions progressively decreased from day +15. At higher MPB/CB ratios, MPB cells had a more important contribution to both early and late engraftment, with the highest cell ratio resulting in only marginal CB cell engraftment. Therefore, our results showed greater potential, on a per cell basis, of human CB vs MPB cells for competitive sustained engraftment in the xenogeneic model used, which was only abrogated by the co-infusion of very high numbers of MPB cells.

Sustained Long Term Engraftment and Transgene Expression of Peripheral Blood CD34+ Cells Transduced with Advanced Lentiviral Vectors in the Preence of ABC Inhibitors.

Because mobilized peripheral blood (MPB) represents an attractive source of cells for gene therapy, we investigated the ability of advanced-generation lentiviral vectors (LV) to transfer the Enhanced Green Fluorescent Protein (EGFP) gene into CD34+ cells isolated from MPB in culture conditions that allowed expansion of transplantable human HSC. So far, a few studies reported transduction of MPB cells with LV pseudotyped with VSV-G. However, the critical issue remains whether primitive, hematopoietic repopulating cells have indeed been transduced.In vitro and in vivo experiments provided here show that MPB CD34+ cells can be effectively transduced by advanced generation LV: they do not lose their proliferation potential, self renewal and multilineage differentiation ability, moreover transgene expression is maintained. Following 48 hour-exposure to FL+TPO+SCF+IL6 and overnight incubation with vector particles, MPB CD34+ cells were further cultured for up to six weeks. During this period of culture the CD34+ cells population, the CD34+/GFP+ fraction and the CFC output were maintained.Transduction efficiency of NOD/SCID repopulating cells (SRC) was assessed by serial transplants into NOD/SCID mice. Primary mice transplanted with transduced cells showed high level of engraftment (11,5 % CD45+ cells): within the CD45+ cells 11,4% expressed GFP (n=15). Moreover, mice transplanted with transduced, week-1 expanded cells showed higher levels of human engraftment than those transplanted with transduced, non-expanded cells (range from 9.25 % to 10.5 % for transduced basal cells; range from 14, 3 % to 38,19 % for transduced 1-week expanded cells). Engraftment was multilineage, with GFP+/lineage+ cells. Serial transplants were performed with transduced, week 1-expanded cells. Secondary engraftment levels were 1.6 %; 19.3 % of human cells were GFP+ (n=3). Moreover, levels of human engraftment were higher in mice injected with transduced, 1- week expanded cells (3.5 %) rather than in those injected with transduced, non expanded cells (1.2 %). Engraftment was multilineage with GFP+/lINEAGE+ cells.These results show that LV efficiently transduced HSC, that maintain proliferation and self-renewal ability.

Treatment of circulating CD34(+) cells with SDF-1alpha or anti-CXCR4 antibody enhances migration and NOD/SCID repopulating potential.

Stromal cell-derived factor-1alpha (SDF-1alpha) has been implicated in homing and engraftment of primitive hematopoietic progenitor cells (HPC) in studies demonstrating reduced NOD/SCID repopulating potential of HPC exposed to supra-physiologic concentrations of SDF-1alpha or anti-CXCR4. Outcome of CXCR4 signaling in some cells has been shown to be dependent on the concentration of SDF-1alpha. We aimed to determine whether similar concentration-dependent responses to CXCR4 signaling are present in CD34(+)cells. Human peripheral blood (PB), mobilized PB (MPB), or bone marrow (BM) CD34(+) cells were incubated for 30 minutes with different concentrations of SDF-1alpha or anti-CXCR4, washed, then assessed for in vitro hematopoietic potential, migration, and NOD/SCID repopulating potential. Exposure of MPB or PB CD34(+) cells to 100 ng/mL SDF-1alpha increased tyrosine phosphorylation without subsequent proliferation or apoptosis. Spontaneous and SDF-1alpha-directed migration also increased in pretreated cells, despite previous exposure to SDF-1alpha. Cells exposed to 1 microg anti-CXCR4/10(6) cells displayed similar increases in activation and migration as cells exposed to SDF-1alpha, demonstrating the ability of anti-CXCR4 to activate the CXCR4 receptor. Interestingly, chimerism in NOD/SCID mice transplanted with MPB CD34(+) cells pretreated with SDF-1alpha or anti-CXCR4 was increased, while exposure of these cells to 10- to 100-fold higher concentrations of these proteins inhibited in vitro migration and NOD/SCID repopulating potential. Migration and NOD/SCID repopulating potential of BM CD34(+) cells remained unchanged after treatment with either protein. These results illustrate the ability of SDF-1alpha and anti-CXCR4 to augment repopulating potential of CD34(+) cells, and suggest that HPC function can be favorably modulated through specific CXCR4 signaling.

Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver

The rate of reconstitution following hematopoietic stem cell (HSC) transplantation differs widely depending on the tissue source of the cells infused. To test the hypothesis that variability in engraftment kinetics is related to differences in the efficiency with which intravenously transplanted HSCs "home" to the bone marrow (BM), the homing properties of murine fetal liver (FL), adult BM, and mobilized peripheral blood (MPB) cells were compared. Lethally irradiated mice transplanted with 2 x 10(6) FL, BM, or MPB cells exhibited sequentially slower recovery of circulating leukocytes and platelets that correlates with the progressively lower frequency of colony-forming cells (CFCs) in these tissues. However, differences in the rate and degree of early and long-term reconstitution were maintained even after infusing equal numbers of CFCs derived from FL, BM, and MPB. To compare the homing of progenitors from these tissues, cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. Three hours later, PKH26(+) cells were reisolated from the BM and spleen by fluorescence-activated cell sorting and assayed for in vitro CFCs. Despite the higher level of very late antigen (VLA)-2, VLA-4, and VLA-5 on Sca-1(+)c-kit(+) cells from FL compared to BM, 10-fold fewer FL CFCs homed to hematopoietic organs than those from BM. MPB cells homed slightly better, but still less efficiently than BM cells. Therefore, clonogenic cells from different tissues exhibit striking variations in homing efficiency that does not necessarily correlate with engraftment kinetics. Homing is likely counterbalanced by intrinsic differences in proliferative potential that ultimately determine the rate of hematopoietic reconstitution.

Investigation into an engraftment defect induced by culturing primitive hematopoietic cells with cytokines

Strategies for transplanting primitive hematopoietic progenitor (PHP) cells are under development that require in vitro manipulation of cells for several hours to several days prior to transplantation. This applies to gene-therapy protocols involving transduction with adenoviral or lentiviral vectors (typically 1 day of ex vivo culture) or retroviral vectors (up to 3 days of culture). Human mobilized peripheral blood (MPB) CD34(+) cells were cultured with the cytokines thrombopoietin mimetic peptide (mTPO), flt3 ligand (FL), and c-kit ligand (KL). Equal numbers of CD34(+) cells, either uncultured or cultured for various time periods up to 5 days, were tested for engraftment in sublethally irradiated 8-10 week-old NOD/SCID mice. Cells were also compared for expression and function of several key surface molecules. At a limiting dose of 1 million cells, mice receiving uncultured cells had a mean of 20% CD45(+) (human) cells in their BM 6 weeks post-transplantation, versus 3% for mice receiving 3-5 day cultured cells. Analysis of 10 surface molecules, CD11a, CD18, CD29, CD49d, CD49e, CXCR-4, CD62L, CD31, CD43, and CD44 over a 5-day culture period showed that their expression levels were either maintained or up-regulated on CD34(+) cells and the primitive Thy-1(+) subset. Similar percentages of uncultured and 3-day cultured MPB CD34(+) cells bound to plates coated with vascular cell adhesion molecule-1 (VCAM-1) under both static and physiological flow conditions, and chemotaxis of cultured cells towards stromal-derived factor-1 (SDF-1) was not impaired, suggesting that VLA-4 and CXCR-4 were functional on cultured cells. CD34(+) Thy-1(+) MPB cells cultured with cytokines expressed increasing levels of Fas receptor beginning at 20 h in culture, with peak expression levels after 3 days (mean Day 0 expression, 39%; mean Day 3 expression, 86%), without increased apoptosis. Including inhibitors of caspases in the media of cells cultured for 24-48 h significantly improved their engraftment in a SCID-hu bone-engraftment model. Increased susceptibility to apoptosis upon in vivo injection may contribute to impaired engraftment of in vitro manipulated cells. Inhibitors of apoptosis may increase their engrafting capacity in clinical settings.

Use of merocyanine 540 for the isolation of quiescent, primitive human bone marrow hematopoietic progenitor cells.

Merocyanine 540 (MC540) is a membrane probe that inserts preferentially into loosely packed domains in the phospholipid bilayer of intact cells. Previous experiments have demonstrated that MC540 will bind to human bone marrow (BM) hematopoietic progenitor cells (HPC). Fractions of mononuclear BM cells expressing high MC540 fluorescence have been shown to be enriched for myeloid progenitors and cells residing in the S/G2 + M phases of the cell cycle. We rationalized that MC540 uptake could be used to distinguish between quiescent and metabolically active cells and, therefore, to fractionate normal and leukemic BM cells and normal mobilized peripheral blood (MPB) cells into functionally distinct groups of progenitors. BM and MPB cells were separated into fractions ranging in fluorescence from MC540Bright to MC540Dim. Cell cycle analysis of these fractions revealed that the MC540Dim fraction of normal and CML BM CD34+ cells constituted the most quiescent fraction, and the MC540Bright fractions from these cell types contained the most actively cycling cells. However, no differences in the percentage of cells in G/G1 were observed between MC540Bright and MC540Dim fractions of MPB CD34+ cells. To investigate if these cell cycle status differences translated into distinct functional properties, the hematopoietic potential of BM CD34+MC540Bright and CD34+MC540Dim cell fractions was analyzed in vitro in long-term BM cultures and limiting dilution analysis (LDA) assays. CD34+MC540Dim cells produced more total and committed progenitor cells in long-term cultures than did the CD34+MC540Bright fraction. The CD34+MC540Dim fraction also contained a 2-fold higher number of long-term hematopoietic culture-initiating cells (LTHCIC) than the CD34+MC540Bright fraction, as defined by LDA assays. These data demonstrate that MC540 can be a useful probe for the isolation of primitive HPC from some hematopoietic tissues and may assist in monitoring structural changes in the phospholipid bilayer during proliferation and differentiation of HPC.

Flt3 Ligand Synergizes With Granulocyte-Macrophage Colony-Stimulating Factor or Granulocyte Colony-Stimulating Factor to Mobilize Hematopoietic Progenitor Cells Into the Peripheral Blood of Mice

AbstractPeripheral blood progenitor cells (PBPC) are increasingly being used in the clinic as a replacement for bone marrow (BM) in the transplantation setting. We investigated the capacity of several different growth factors, including human flt3 ligand (FL), alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF ) or granulocyte colony-stimulating factor (G-CSF ), to mobilize colony forming cells (CFU) into the peripheral blood (PB) of mice. Mice were injected subcutaneously (SC) with growth factors daily for up to 10 days. Comparing the single agents, we found that FL alone was superior to GM-CSF or G-CSF in mobilizing CFU into the PB. FL synergized with both GM-CSF or G-CSF to mobilize more CFU, and in a shorter period of time, than did any single agent. Administration of FL plus G-CSF for 6 days resulted in a 1,423-fold and 2,717-fold increase of colony-forming unit–granulocyte-macrophage (CFU-GM) and colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) in PB, respectively, when compared with control mice. We also followed the kinetics of CFU numerical changes in the BM of mice treated with growth factors. While GM-CSF and G-CSF alone had little effect on BM CFU over time, FL alone increased CFU-GM and CFU-GEMM threefold and fivefold, respectively. Addition of GM-CSF or G-CSF to FL did not increase CFU in BM over levels seen with FL alone. However, after the initial increase in BM CFU after FL plus G-CSF treatment for 3 days, BM CFU returned to control levels after 5 days treatment, and CFU-GM were significantly reduced (65%) after 7 days treatment, when compared with control mice. Finally, we found that transplantation of FL or FL plus G-CSF–mobilized PB cells protected lethally irradiated mice and resulted in long-term multilineage hematopoietic reconstitution.