Production Of Hematopoietic Cells Research Articles

HDAC1 and HDAC2 Modulate TGF-β Signaling during Endothelial-to-Hematopoietic Transition.

SummaryThe first hematopoietic stem and progenitor cells are generated during development from hemogenic endothelium (HE) through trans-differentiation. The molecular mechanisms underlying this endothelial-to-hematopoietic transition (EHT) remain poorly understood. Here, we explored the role of the epigenetic regulators HDAC1 and HDAC2 in the emergence of these first blood cells in vitro and in vivo. Loss of either of these epigenetic silencers through conditional genetic deletion reduced hematopoietic transition from HE, while combined deletion was incompatible with blood generation. We investigated the molecular basis of HDAC1 and HDAC2 requirement and identified TGF-β signaling as one of the pathways controlled by HDAC1 and HDAC2. Accordingly, we experimentally demonstrated that activation of this pathway in HE cells reinforces hematopoietic development. Altogether, our results establish that HDAC1 and HDAC2 modulate TGF-β signaling and suggest that stimulation of this pathway in HE cells would be beneficial for production of hematopoietic cells for regenerative therapies.

RENIN-ANGIOTENSIN SYSTEM IN REGULATION OF HEMATOPOIESIS

The renin-angiotensin system (RAS) has long been known as the endocrine system involved in the regulation of arterial pressure and waterelectrolyte balance. Local (tissue) RAS can influence cellular activity, tissue damage and regeneration. In the bone marrow there are active ligands of peptides, mediators, receptors and signaling pathways of the RAS. Local RAS can influence the growth, production, proliferation and differentiation of hematopoietic cells and participate in the regulation of both normal and pathological hematopoiesis. Angiotensin-converting enzyme (ACE) CD143 plays a key role in the classical RAS. After differentiation from hemangioblast, hematopoietic progenitor cells constantly express ACE in human embryonic, fetal and adult hematopoietic tissues, as well as at all stages of hematopoietic ontogeny. The ACE cleaves the C-terminal dipeptide and thus forms the octapeptide Angiotensin II. In addition to angiotensin II, ACE also regulates a group of biologically active peptides, such as substance P, ac-SDKP and angiotensin 1–7. Local RAS is also one of the most important components in the tumor microenvironment, affecting tumor growth and metastasis by autocrine and paracrine pathways, modulating numerous carcinogenic events such as angiogenesis, apoptosis, cell proliferation, immune responses, and extracellular matrix formation. The purpose of this review is to describe the known functions of local RAS in the hematopoiesis regulation. More detailed study of the RAS components mechanisms of action will expand therapy approaches in the neoplastic diseases and in bone marrow transplantation.

386 - Trypan Blue Viability as an Alternative to CD34-Specific Viability for Frozen-Thawed Hematopoietic Progenitor Cell Products to Meet FACT and AABB Standards

386 - Trypan Blue Viability as an Alternative to CD34-Specific Viability for Frozen-Thawed Hematopoietic Progenitor Cell Products to Meet FACT and AABB Standards

Megakaryocyte-derived excessive transforming growth factor β1 inhibits proliferation of normal hematopoietic stem cells in acute myeloid leukemia

Impaired production of healthy hematopoietic cells from residual hematopoietic stem cells (HSCs) leads to high mortality in acute myeloid leukemia (AML). Previous studies have identified p21 and Egr3 as intrinsic factors responsible for the growth arrest and differentiation blockade of normal HSCs in leukemia; however, the related extrinsic factors remain unknown. In this study, we found that transforming growth factor β (TGFβ) signaling was upregulated in HSCs from bone marrow of mice with MLL-AF9-induced acute myeloid leukemia (AML) because of excessive production of TGFβ1, especially from megakaryocytes, and overactivation of latent TGFβ1 protein. We also found that SMAD3, a signal transducer of TGFβ1, directly bound to Egr3 and upregulated its expression to arrest proliferation of HSCs. Our study provides evidence for targeting TGFβ1 in AML to rectify normal hematopoiesis defects in clinical practice.

The mirn23a and mirn23b microrna clusters are necessary for proper hematopoietic progenitor cell production and differentiation

Mice deficient for microRNA (miRNA) cluster mirn23a exhibit increased B lymphopoiesis at the expense of myelopoiesis, whereas hematopoietic stem and progenitor cell (HSPC) populations are unchanged. Mammals possess a paralogous mirn23b gene that can give rise to three mature miRNAs (miR-23b, miR-24-1, and miR-27b) that have identical seed/mRNA-targeting sequences to their mirn23a counterparts. To assess whether compound deletion of mirn23a and mirn23b exacerbates the hematopoietic phenotype observed in mirn23a-/- mice, we generated a compound mirn23a-/-mirn23bfl/fl:Mx1-Cre conditional knockout mouse and assayed hematopoietic development after excision of mirn23b. Loss of both genes in adult bone marrow further skewed HSPC differentiation toward B cells at the expense of myeloid cells, demonstrating a dosage-dependent effect on regulating cell differentiation. Strikingly, double-knockout (DKO) mice had decreased bone marrow cellularity with significantly decreased hematopoietic stem cell and HSPC populations, a phenotype not observed in mice deficient for mirn23a alone. Competitive transplantation assays showed decreased contribution of mirn23a-/-mirn23b-/- HSPCs to hematopoietic lineages at 6 and 12 weeks after transplantation. Defects in the proliferation of mirn23a-/-b-/- HSPCs was not observed; however, DKO cells were more apoptotic compared with both wild-type and mirn23a-/- cells. Together, our data show that complete loss of mirn23a/mirn23b miRNAs results in decreased blood production and affects lineage output in a concentration-dependent manner.

Single Cell Resolution of Human Hematoendothelial Cells Defines Transcriptional Signatures of Hemogenic Endothelium.

Endothelial-to-hematopoietic transition (EHT) is an important stage in definitive hematopoietic development. However, the genetic mechanisms underlying human EHT remain poorly characterized. We performed single cell RNA-seq using 55 hemogenic endothelial cells (HECs: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c+ ), 47 vascular endothelial cells without hematopoietic potential (non-HE: CD31+ CD144+ CD41- CD43- CD45- CD73- RUNX1c- ), and 35 hematopoietic progenitor cells (HPCs: CD34+ CD43+ RUNX1c+ ) derived from human embryonic stem cells (hESCs). HE and HP were enriched in genes implicated in hemogenic endothelial transcriptional networks, such as ERG, GATA2, and FLI. We found transcriptional overlap between individual HECs and HPCs; however, these populations were distinct from non-HE. Further analysis revealed novel biomarkers for human HEC/HPCs, including TIMP3, ESAM, RHOJ, and DLL4. Collectively, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE are more heterogeneous and transcriptionally distinct. Our findings provide a novel strategy to test new genetic targets and optimize the production of definitive hematopoietic cells from human pluripotent stem cells. Stem Cells 2018;36:206-217.

Phase 2 Trials with Mgta-456, Single Cord Blood Units (CBU) Expanded with an Aryl Hydrocarbon Receptor (AHR) Antagonist, Demonstrate Uniform Engraftment and Rapid Hematopoietic Recovery in Patients Following Myeloablative or Non-Myeloablative Conditioning

Background. The use of umbilical cord blood (UCB) in transplant has been limited by the low number of CD34+ cells, resulting in prolonged periods of cytopenia for patients and high risk of graft failure, thereby restricting its widespread application. MGTA-456 is the hematopoietic cell product after cord blood CD34+ cells are placed in expansion culture for 15 days with an aryl hydrocarbon receptor (AHR) antagonist in the presence of SCF, Flt-3L, IL-6 and TPO. In a prior Phase 1/2 safety study, 18 patients received MGTA-456, its accompanying CD34negfraction and a larger, unexpanded UCB unit. All patients engrafted at a median of 14.5 days (range, 7-23), significantly faster than similarly treated historical controls (p<0.01). Based on these results, two Phase 2 studies were initiated to evaluate the effectiveness of MGTA-456 as a stand-alone graft after myeloablative conditioning (MAC) or non-myeloablative conditioning (NMAC).Patients and Methods : Twenty patients with high-risk hematologic malignancy and a partially HLA-matched CBU were enrolled; 10 were treated with cyclophosphamide (CY) 120 mg/kg, fludarabine (FLU) 75 mg/m2 and total body irradiation (TBI) 1320 cGy (MAC) and 10 with CY 50 mg/kg, FLU 200 mg/m2 and TBI 200 cGy (NMAC). All patients received cyclosporine and mycophenolate mofetil post-transplant immunoprophylaxis. Expansion was low in 2 UCB units, therefore 18 patients received MGTA-456 and its CD34neg fraction.Results : Expansion culture yielded a median of 1,227 x 106 CD34+ cells (range, 201-8969) as compared to the input number of 4.2 x 106 (range, 1.4-16.3) after CD34 selection - a 324-fold (range, 42-1643) expansion of CD34+ cells. As transplant results vary by intensity of the conditioning, patient outcomes were compared to similarly treated historical cohorts between 2006 and 2015 (n=151 MAC; n=132 NMAC). For both groups, demographics were similar except for more recent year of transplant for recipients of MGTA-456. For recipients of MAC, MGTA-456 engrafted in all patients at a median of 14 days (range, 7-32) as compared to 89% engraftment at a median of 23 days (range, 19-31) in the control population (p<0.01, see Figure 1). Complete chimerism was rapid for both myeloid and T cells with no late graft failures; the longest follow-up was 5.6 years in recipients of MGTA-456. For recipients of NMAC, MGTA-456 also engrafted in all patients at a median of 7 days (range, 6-14) as compared to 94% engraftment at a median of 15 days (range, 7-22). In contrast to complete chimerism seen after MAC, chimerism is often mixed for the first month in both myeloid and T cells after NMAC. Compared to the historical cohort, recipients of MGTA-456 had more rapid chimerism after NMAC. CD34 cell dose correlates with speed of recovery but only in recipients with MAC; in recipients of NMAC, recovery is uniformly rapid regardless of CD34 cell dose. Additionally, immune recovery as measured by an absolute CD4 count >200/uL was achieved at day 60 (median) in recipients of MGTA-456 regardless of conditioning regimen. Results were also encouraging for other transplant outcomes. For recipients of MGTA-456 compared to the historical cohort after MAC, incidence of acute GVHD (aGVHD) grade 3-4 was 22% vs 24%; chronic GVHD (cGVHD), 11% vs 21%; transplant-related mortality (TRM), 11% vs 34%; and overall survival (OS), 67% vs 55%. After NMAC, results were similar between cohorts except for a higher risk of aGVHD in recipients of MGTA-456 (aGVHD 3-4, 43% vs 15%; cGVHD, 0% vs 19%; TRM, 22% vs 20%; and OS, 44% vs 49%). The increased rate of aGVHD in the NMAC cohort likely reflects non-compliance with prescribed GVHD immunoprophylaxis in 2 of 9 recipients.Conclusion : In these studies, MGTA-456 significantly accelerated hematopoietic recovery and abrogated the engraftment barrier typically associated with UCB transplantation. The marked expansion of CD34+ cells in MGTA-456 suggests that a significant number of patients will have an adequate single CBU and better HLA matched graft since a greater proportion of the cord blood inventory will be available irrespective of weight. Given these promising results, additional studies are being planned. [Display omitted] DisclosuresWagner:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Brunstein:Novartis: Research Funding; Magenta Therapeutics: Research Funding. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties; Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. McKenna:Magenta Therapeutics: Research Funding; Novartis: Research Funding. Sanna:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Bleul:Hoffmann-La Roche AG: Employment. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Hematological alterations in protein malnutrition.

Protein malnutrition is one of the most serious nutritional problems worldwide, affecting 794 million people and costing up to $3.5 trillion annually in the global economy. Protein malnutrition primarily affects children, the elderly, and hospitalized patients. Different degrees of protein deficiency lead to a broad spectrum of signs and symptoms of protein malnutrition, especially in organs in which the hematopoietic system is characterized by a high rate of protein turnover and, consequently, a high rate of protein renewal and cellular proliferation. Here, the current scientific information about protein malnutrition and its effects on the hematopoietic process is reviewed. The production of hematopoietic cells is described, with special attention given to the hematopoietic microenvironment and the development of stem cells. Advances in the study of hematopoiesis in protein malnutrition are also summarized. Studies of protein malnutrition in vitro, in animal models, and in humans demonstrate several alterations that impair hematopoiesis, such as structural changes in the extracellular matrix, the hematopoietic stem cell niche, the spleen, the thymus, and bone marrow stromal cells; changes in mesenchymal and hematopoietic stem cells; increased autophagy; G0/G1 cell-cycle arrest of progenitor hematopoietic cells; and functional alterations in leukocytes. Structural and cellular changes of the hematopoietic microenvironment in protein malnutrition contribute to bone marrow atrophy and nonestablishment of hematopoietic stem cells, resulting in impaired homeostasis and an impaired immune response.

Preclinical studies for a phase 1 clinical trial of autologous hematopoietic stem cell gene therapy for sickle cell disease

Background aimsGene therapy by autologous hematopoietic stem cell transplantation (HSCT) represents a new approach to treat sickle cell disease (SCD). Optimization of the manufacture, characterization and testing of the transduced hematopoietic stem cell final cell product (FCP), as well as an in depth in vivo toxicology study, are critical for advancing this approach to clinical trials. MethodsData are shown to evaluate and establish the feasibility of isolating, transducing with the Lenti/βAS3-FB vector and cryopreserving CD34+ cells from human bone marrow (BM) at clinical scale. In vitro and in vivo characterization of the FCP was performed, showing that all the release criteria were successfully met. In vivo toxicology studies were conducted to evaluate potential toxicity of the Lenti/βAS3-FB LV in the context of a murine BM transplant. ResultsPrimary and secondary transplantation did not reveal any toxicity from the lentiviral vector. Additionally, vector integration site analysis of murine and human BM cells did not show any clonal skewing caused by insertion of the Lenti/βAS3-FB vector in cells from primary and secondary transplanted mice. ConclusionsWe present here a complete protocol, thoroughly optimized to manufacture, characterize and establish safety of a FCP for gene therapy of SCD.

Forme pseudo-tumorale de la tuberculose osseuse chez l’enfant : à propos d’une localisation humérale rare

Impaired production of healthy hematopoietic cells from residual hematopoietic stem cells (HSCs) leads to high mortality in acute myeloid leukemia (AML). Previous studies have identified p21 and Egr3 as intrinsic factors responsible for the growth arrest and differentiation blockade of normal HSCs in leukemia; however, the related extrinsic factors remain unknown. In this study, we found that transforming growth factor β (TGFβ) signaling was upregulated in HSCs from bone marrow of mice with MLL-AF9-induced acute myeloid leukemia (AML) because of excessive production of TGFβ1, especially from megakaryocytes, and overactivation of latent TGFβ1 protein. We also found that SMAD3, a signal transducer of TGFβ1, directly bound to Egr3 and upregulated its expression to arrest proliferation of HSCs. Our study provides evidence for targeting TGFβ1 in AML to rectify normal hematopoiesis defects in clinical practice.

MRNA-mediated glycoengineering ameliorates deficient homing of human stem cell-derived hematopoietic progenitors.

Generation of functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) has been a long-sought-after goal for use in hematopoietic cell production, disease modeling, and eventually transplantation medicine. Homing of HSPCs from bloodstream to bone marrow (BM) is an important aspect of HSPC biology that has remained unaddressed in efforts to derive functional HSPCs from human PSCs. We have therefore examined the BM homing properties of human induced pluripotent stem cell-derived HSPCs (hiPS-HSPCs). We found that they express molecular effectors of BM extravasation, such as the chemokine receptor CXCR4 and the integrin dimer VLA-4, but lack expression of E-selectin ligands that program HSPC trafficking to BM. To overcome this deficiency, we expressed human fucosyltransferase 6 using modified mRNA. Expression of fucosyltransferase 6 resulted in marked increases in levels of cell surface E-selectin ligands. The glycoengineered cells exhibited enhanced tethering and rolling interactions on E-selectin-bearing endothelium under flow conditions in vitro as well as increased BM trafficking and extravasation when transplanted into mice. However, glycoengineered hiPS-HSPCs did not engraft long-term, indicating that additional functional deficiencies exist in these cells. Our results suggest that strategies toward increasing E-selectin ligand expression could be applicable as part of a multifaceted approach to optimize the production of HSPCs from human PSCs.

Thrombopoietin contributes to the formation and the maintenance of hematopoietic progenitor-containing cell clusters in the aorta-gonad-mesonephros region

In the midgestation mouse embryo, hematopoietic cell clusters containing hematopoietic stem/progenitor cells arise in the aorta-gonad-mesonephros (AGM) region. We have previously reported that forced expression of the Sox17 transcription factor in CD45lowc-Kithigh AGM cells, which are the hematopoietic cellular component of the cell clusters, and subsequent coculture with OP9 stromal cells in the presence of three cytokines, stem cell factor (SCF), interleukin-3 (IL-3), and thrombopoietin (TPO), led to the formation and the maintenance of cell clusters with cells at an undifferentiated state in vitro. In this study, we investigated the role of each cytokine in the formation of hematopoietic cell clusters. We cultured Sox17-transduced AGM cells with each of the 7 possible combinations of the three cytokines. The size and the number of Sox17-transduced cell clusters in the presence of TPO, either alone or in combination, were comparable to that observed with the complete set of the three cytokines. Expression of TPO receptor, c-Mpl was almost ubiquitously expressed and maintained in Sox17-transduced hematopoietic cell clusters. In addition, the expression level of c-Mpl was highest in the CD45lowc-Kithigh cells among the Sox17-transduced cell clusters. Moreover, c-Mpl protein was highly expressed in the intra-aortic hematopoietic cell clusters in comparison with endothelial cells of dorsal aorta. Finally, stimulation of the endothelial cells prepared from the AGM region by TPO induced the production of hematopoietic cells. These results suggest that TPO contributes to the formation and the maintenance of hematopoietic cell clusters in the AGM region.

Efficient Production of Human Hematopoietic Cells from Pluripotent Stem Cells through cAMP Induction

Efficient Production of Human Hematopoietic Cells from Pluripotent Stem Cells through cAMP Induction

Single-Cell Resolution of Human Pluripotent Stem Cell Derived Hemato-Endothelial Cells Reveals Distinct Transcriptional Signatures of Hemogenic Endothelium

Hemogenic endothelium is a rare and highly specialized subset of vascular endothelial cell that functions as a precursor cell population to definitive blood development. One of the mechanistic hallmarks of definitive hematopoiesis is the endothelial-to-hematopoietic transition (EHT), a process where hemogenic endothelial cells phenotypically switches to produce a detached and free-moving hematopoietic cell. While EHT has been visualized both in vitro and in vivo via lineage tracing studies, the regulation of this fate change at both a cellular and molecular level remains unclear. Human pluripotent stem cells, such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can serve as a useful platform to characterize human hemogenic endothelium and to understand basic mechanisms underlying human EHT. We hypothesized that human hemogenic endothelium derived from hESCs is phenotypically and transcriptionally distinct from other vascular endothelial cells and hematopoietic progenitor cells. To identify human hemogenic endothelium, we used combined expression of endothelial cell surface antigens and RUNX1c expression via a fluorescent reporter previously validated in our lab. Specifically, we employed defined culture methods to differentiate hemogenic endothelial cells (HE) and vascular endothelial cells without hematopoietic potential (non-HE) from hESC harboring a RUNX1c-tdTomato reporter (hESC-RUNX1c-tdTomato). At Day 11 of differentiation, CD31+CD144+ endothelial cells were present, with approximately 40% of these cells tdTomato+. We next sorted HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato+) and non-HE (CD31+CD144+CD41-CD43-CD45-CD73-tdTomato-) and cultured both populations in endothelial growth media (EGM) or hematopoietic growth media. HE retained characteristic cobblestone morphology and CD31 expression over the course of 5 days in EGM similar to control human umbilical vein endothelial cells. HE, but not non-HE, was able to generate non-adherent, tdTomato+ hematopoietic progenitor cells in hematopoietic growth media. Using these defined cell populations, we next performed single-cell RNASeq on HE, non-HE, and early hematopoietic progenitor cells (HP; CD34+CD43+tdTomato+). We captured a total of 55 HE, 47 non-HE, and 35 HP using the Fluidgm C1 single cell system and performed next generation sequencing of validated libraries. We analyzed single cell gene expression using Seurat, an R-based bioinformatics software package developed for the analysis of single cell NGS experiments. Populations were first validated based on expression of known genetic identifiers for vascular endothelium, hemogenic endothelium, and hematopoietic progenitor cells. HE and HP were both highly enriched for CDH5, ERG, ESAM, and FLI as compared to non-HE; these genes have been previously implicated in HE functionality. All single cell transcriptional profiles were similarly enriched for KDR, PECAM1, and LMO2, which are characteristic genes expressed in vascular endothelium. We next performed t-distributed stochastic neighbor embedding (t-SNE) using statistically significant principal components to distinguish groups of cells with similar transcriptional expression. Interestingly, we found overlap between individual HE and HP cells (Cluster 1); however, these cells were distinct from two, separate clusters of non-HE (Cluster 2 & 3; Figure 1). Further analysis of these clusters revealed novel biomarkers for HE/HP, such as TIMP3 (DGE: 2.06, Power: 0.91), ERG (DGE: 2.08, Power: 0.83), NOTCH4 (DGE: 2.35, Power: 0.78), and HEY1 (DGE: 2.47, Power: 0.74), Non-HE were found to cluster into two, distinct groups (Clusters 2 and 3), with Cluster 3 enriched for extracellular matrix genes (COL1A1 (DGE: 6.58, Power: 1.00), DCN (DGE: 6.09, Power: 1.00), VCAN (DGE: 3.55, Power: 1.00), FN1 (DGE: 2.19, Power: 0.96)). This profile suggests that some hESC-derived non-HE further differentiate into mesenchymal cells, in a process known as the endothelial-to mesenchymal transition (EndMT). Taken together, we demonstrate that hESC-derived HE and HP share a common developmental pathway, while non-HE is heterogeneous, but transcriptionally distinct. Our novel findings will be instrumental for testing new genetic targets to optimize the production of definitive hematopoietic cells. [Display omitted] DisclosuresKaufman:Fate Therapeutics: Consultancy.

NaïVe T Cell Depletion of PBSC Grafts Results in Very Low Rates of Chronic Gvhd and High Survival

BackgroundGraft-versus-host disease (GVHD) frequently causes morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT) as a result of organ damage and infections. In HLA-identical HCT, GVHD results from recognition by donor T cells of minor histocompatibility (H) antigens on recipient tissues. Complete T cell depletion (TCD) of donor hematopoietic cell products is more effective than pharmacologic immunosuppression for preventing GVHD, but is complicated by delayed immune reconstitution and consequent life-threatening infections.Approaches to HCT which preferentially deplete the T cells that primarily cause GVHD and preserve pathogen-specific T cells may improve HCT outcomes.Mature CD3+ CD8+ and CD3+ CD4+ T cells can be classified into CD45RA+ CD62L+ naïve (TN) and CD45RO+ memory (TM) subsets, the latter of which includes effector memory (TEM) and central memory (TCM) cells. Murine studies in which allogeneic TCD bone marrow (BM) is transplanted with purified T cells from individual T cell subsets to irradiated minor H antigen disparate recipients have demonstrated that the most severe GVHD results from transplanting T cells of the TN subset. Purified TCM causes mild GVHD and TEM do not cause detectable GVHD and can transfer immunity to pathogens.In vitro studies have similarly demonstrated that human donor CD8+ T cells specific for recipient minor H antigens are found predominantly within the TN cell subset, suggesting selective TN cell depletion may alter the GVHD incidence and/or severity in human HCT.Methods and resultsWe developed an effective process for engineering human peripheral blood stem cell (PBSC) grafts that depletes CD45RA+ TN cells and retains CD34+ stem cells and functional CD45RO+ TM cells specific for a broad range of opportunistic pathogens (Bleakley BBMT 2014). We are conducting clinical trials to evaluate the selective depletion of TN cells from HLA-matched allogeneic PBSC grafts for the prevention of GVHD in patients with acute leukemia, the first of which has been published (Bleakley JCI 2015, N=35). Seventy patients have now been treated on three consecutive phase II trials. The median age was 34 years (1-56 years), 56% of patients had a diagnosis of ALL, 46% had previously relapsed or had detectable disease (MRD or relapse) at the time of HCT, and 23% had unrelated donor (URD) grafts. Intensive myeloablative, TBI-containing (13.2Gy) conditioning was used for 63 patients, whilst 7 patients received a medium intensity ’midi’ preparative regimen, including 4Gy of TBI. The TN-depletion procedure was successfully performed on URD PBSC products shipped overnight from donor centers throughout the US, as well as on MRD PBSC collected at our centers. Reliable engraftment with high-level donor chimerism was observed in recipients of ’midi’ as well as intensive myeloablative conditioning. The 2-year estimates of overall survival, disease-free survival, survival free of relapse and chronic GVHD (CRFS) and survival free of relapse, grade II-IV acute GVHD, and chronic GVHD (GRFS) are 79%, 73%, 69% and 63% respectively. Median follow-up among survivors is 26 months. The frequency and severity of chronic GVHD is remarkably low (5%) compared to historical rates of 40-60% chronic GVHD in HLA-matched PBSC transplantation with conventional calcineurin inhibitor-based immunosuppression. Relapse and non-relapse mortality (NRM) are acceptably low at 19% and 8%, respectively. No NRM occurred in patients <40 years. Updated results will be presented.ConclusionsThe outcomes of recipients of TN-depleted PBSC grafts compare very favorably to published results of HCT for patients with acute leukemia. For example, the 69% incidence of CRFS at 2 years in TN-depleted recipients compares with reported 2-year GRFS rates of 37% and 17% in recipients of allogeneic PBSC from HLA-matched related donors with or without ATG (Kroger et al. NEJM 2016). Our results suggest that TN-depletion of PBSC grafts may reduce the risk of chronic GVHD without negatively impacting other important HCT outcomes. DisclosuresRiddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Cell Medica: Consultancy, Honoraria; Adaptive Biotechnologies: Consultancy, Honoraria.

Evolution of malignant plasmacytoma cell lines from K14E7 Fancd2-/- mouse long-term bone marrow cultures.

We tested the effect of expression of the Human Papilloma Virus (HPV E7) oncogene on hematopoiesis in long-term bone marrow cultures (LTBMCs) derived from K14E7 (FVB) Fancd2−/− (129/Sv), K14E7 Fancd2+/+, Fancd2−/−, and control (FVB X 129/Sv) Fl mice. K14E7 Fancd2−/− and Fancd2−/− LTBMCs showed decreased duration of production of total nonadherent hematopoietic cells and progenitors forming day 7 and day 14 multilineage CFU-GEMM colonies in secondary cultures (7 wks and 8 wks respectively) compared to cultures from K14E7 Fancd2+/+ (17 wks) or control mice (18 wks) p < 0.0001. Marrow stromal cell lines derived from both K14E7 Fancd2−/− and Fancd2−/− cultures were radiosensitive, as were IL-3 dependent hematopoietic progenitor cell lines derived from K14E7 Fancd2−/− cultures. In contrast, Fancd2−/− mouse hematopoietic progenitor cell lines and fresh marrow were radioresistant. K14E7 Fancd2−/− mouse freshly explanted bone marrow expressed no detectable K14 or E7; however, LTBMCs produced K14 positive factor-independent (FI) clonal malignant plasmacytoma forming cell lines in which E7 was detected in the nucleus with p53 and Rb. Transfection of an E6/E7 plasmid into Fancd2−/−, but not control Fancd2+/+ IL-3 dependent hematopoietic progenitor cell lines, increased cloning efficiency, cell growth, and induced malignant cell lines. Therefore, the altered radiobiology of hematopoietic progenitor cells and malignant transformation in vitro by K14E7 expression in cells of the Fancd2−/− genotype suggests a potential role of HPV in hematopoietic malignancies in FA patients.

The influence of protein malnutrition on the production of GM-CSF and M-CSF by macrophages

It is well established that protein malnutrition (PM) impairs immune defenses and increases susceptibility to infection. Macrophages are cells that play a central role in innate immunity, constituting one of the first barriers against infections. Macrophages produce several soluble factors, including cytokines and growth factors, important to the immune response. Among those growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF). GM-CSF and M-CSF are important to monocyte and macrophage development and stimulation of the immune response process. Knowing the importance of GM-CSF and M-CSF, we sought to investigate the influence of PM on macrophage production of these growth factors. Two-month-old male BALB/c mice were subjected to PM with a low-protein diet (2%) and compared to a control diet (12%) mouse group. Nutritional status, hemogram and the number of peritoneal cells were evaluated. Additionally, peritoneal macrophages were cultured and the production of GM-CSF and M-CSF and mRNA expression were evaluated. To determine if PM altered macrophage production of GM-CSF and M-CSF, they were stimulated with TNF-α. The PM animals had anemia, leukopenia and a reduced number of peritoneal cells. The production of M-CSF was not different between groups; however, cells from PM animals, stimulated with or without TNF-α, presented reduced capability to produce GM-CSF. These data imply that PM interferes with the production of GM-CSF, and consequently would affect the production and maturation of hematopoietic cells and the immune response.

High-Efficiency Serum-Free Feeder-Free Erythroid Differentiation of Human Pluripotent Stem Cells Using Small Molecules.

This differentiation protocol allows the production of a large amount of erythroid cells from pluripotent stem cells. Its efficiency is compatible with that of in vitro red blood cell production, and it can be a considerable asset for studying developmental erythropoiesis and red blood cell enucleation, thereby aiding both basic and translational research. In addition to red cells, the early stages of the protocol could also be used as a starting point for the large-scale production of other hematopoietic cell types, including the ultimate goal of generating long-term reconstituting hematopoietic stem cells.

Enforced Expression of HOXB4 in Human Embryonic Stem Cells Enhances the Production of Hematopoietic Progenitors but Has No Effect on the Maturation of Red Blood Cells.

: We have developed a robust, Good Manufacturing Practice-compatible differentiation protocol capable of producing scalable quantities of red blood cells (RBCs) from human pluripotent stem cells (hPSCs). However, translation of this protocol to the clinic has been compromised because the RBCs produced are not fully mature; thus, they express embryonic and fetal, rather than adult globins, and they do not enucleate efficiently. Based on previous studies, we predicted that activation of exogenous HOXB4 would increase the production of hematopoietic progenitor cells (HPCs) from hPSCs and hypothesized that it might also promote the production of more mature, definitive RBCs. Using a tamoxifen-inducible HOXB4-ER(T2) expression system, we first demonstrated that activation of HOXB4 does increase the production of HPCs from hPSCs as determined by colony-forming unit culture activity and the presence of CD43(+)CD34(+) progenitors. Activation of HOXB4 caused a modest, but significant, increase in the proportion of immature CD235a(+)/CD71(+) erythroid cells. However, this did not result in a significant increase in more mature CD235a(+)/CD71(-) cells. RBCs produced in the presence of enhanced HOXB4 activity expressed embryonic (ε) and fetal (γ) but not adult (β) globins, and the proportion of enucleated cells was comparable to that of the control cultures. We conclude that programming with the transcription factor HOXB4 increases the production of hematopoietic progenitors and immature erythroid cells but does not resolve the inherent challenges associated with the production of mature adult-like enucleated RBCs. As worldwide blood donations decrease and transfusable transmitted infections increase, intense interest has ensued in deriving red blood cells (RBCs) in vitro from alternative sources such as pluripotent stem cells. A translatable protocol was developed to generate RBCs; however, these RBCs have an immature phenotype. It was hypothesized that the transcription factor HOXB4 could enhance their production and maturation. Although HOXB4 increased the production of erythroid progenitors, it did not promote their maturation. Despite the remaining challenges, a robust system has been established to test other candidates and add to the knowledge base in this field.

77 - Impact of Mobilization Regimen and Apheresis Instrument on Hematopoietic Progenitor Cell Collection Efficiency and Product Quality

77 - Impact of Mobilization Regimen and Apheresis Instrument on Hematopoietic Progenitor Cell Collection Efficiency and Product Quality