Hematopoietic Stem/precursor Cells Research Articles

Interim Safety and Efficacy Results from a Phase 1/2 Study of Zinc Finger Nuclease-Modified Autologous Hematopoietic Stem Cells for Sickle Cell Disease (PRECIZN-1)

Introduction Sickle cell disease (SCD) is caused by pathologic variants in both alleles of the β-globin gene. Elevated fetal hemoglobin (HbF) levels inhibit sickle cell formation and ameliorate symptoms and improve survival in patients with SCD (Hebert, Am J Hematol, 2020;95:1235-45). BIVV003 is a novel therapeutic product comprising autologous CD34+ hematopoietic stem precursor cells (HSPC) modified ex vivo by zinc finger nucleases (ZFN) targeting specifically the BCL11A gene erythroid-specific enhancer (ESE) to increase endogenous HbF production in erythrocytes (RBC). Methods PRECIZN-1 (NCT03653247) is an ongoing first-in-human, open label, single arm, multi-center study evaluating safety and tolerability of BIVV003 (n=8; aged 18-40 years) with severe SCD. Eligible subjects underwent mobilization and apheresis with plerixafor 240 ug/kg/day for up to 3 days to collect autologous CD34+HSPCs for manufacturing of BIVV003. HSPCs were transfected ex vivo with ZFN mRNAs targeting the ESE region of the BCL11A locus to manufacture BIVV003. After myeloablative conditioning with busulfan 3.2 mg/kg/day intravenously (IV) for 4 days with dose adjustment based on pharmacokinetics, subjects received BIVV003 as a single minimum dose of 3× 106 CD34+ HSPC/kg IV. Subjects were monitored for stem cell engraftment and hematopoietic recovery, adverse events (AEs), clinical and laboratory hemolysis markers, total hemoglobin (Hb) and HbF, RBC containing HbF (F cells), and SCD-related events for up to 104 weeks post-BIVV003 infusion. Results As of the cutoff date of 03 May 2022, 4 subjects have received BIVV003 infusions up to 125 weeks of follow-up. A fifth subject was dosed with BIVV003 on 04 May 2022 and has engrafted. Among the 5 subjects, the male:female ratio is 3:2, the age range is 18-35 years, and 4 have genotype HbSS and 1 HbS-β0. No subject has required HSPC rescue; all subjects had hematopoietic reconstitution without requiring RBC or platelet transfusions for more than one month post-BIVV003 infusion. The average time to neutrophil and platelet engraftment was 23 days and 33 days, respectively. In bone marrow aspirates 26 weeks post-infusion, the frequency of cells containing indels at the BCL11A ESE target locus was in the range of 17.1-34.1%. No clonal dominance has been observed in bone marrow or peripheral blood cells. By Week 26, all 4 subjects reached a protective level of ≥ 10 pg HbF/F cell that inhibits HbS polymerization (Steinberg, Blood, 2014;123:481-485), and this level was sustained in 3 out of 4 subjects through the most recent assessment at Week 104, 78, and 52, respectively, along with F cell levels of 90% or higher. The change in total Hb from baseline (at screening) to the latest visit was -0.3-3.1 g/dL, and the HbF fraction increased to 12.2-41.2% (Figure 1). Three subjects have had no recurrence of severe vaso-occlusive crises (VOCs) post-infusion, compared to experiencing 6 to 22 severe VOCs in the 2 years pre-study. The one subject who did not sustain a HbF/F level ≥ 10 pg experienced 2 severe VOCs at 9 and 16 months post-infusion. The adverse events (AEs) reported were consistent with plerixafor mobilization and busulfan myeloablative conditioning. No AEs related to BIVV003 were reported. Conclusions The interim safety and efficacy results reported here confirm the potential therapeutic value of ZFN-mediated modification of the BCL11A ESE region and BIVV003 infusion to address current unmet needs of patients with SCD. BIVV003 has been well tolerated in the 5 subjects infused to date, with no AEs related to BIVV003. Three of the 4 subjects infused and with over 52 weeks of follow-up have had stable engraftment of ZFN-modified HSPCs resulting in sustained elevated HbF levels and absence of severe VOCs post-BIVV003 administration. A fifth subject received BIVV003 manufactured using an improved process that has shown in internal experiments to increase the number of ZFN-modified long-term progenitors in the drug product. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Definitive hematopoietic stem/progenitor cells from human embryonic stem cells through serum/feeder-free organoid-induced differentiation

BackgroundEx vivo production of hematopoietic stem/precursor cells (HSPCs) represents a promising versatile approach for blood disorders.MethodsTo derive definitive HSPCs from human embryonic stem cells (ESCs), we differentiated mesodermally specified embryoid bodies (EBs) on gelatin-coated plates in serum/feeder-free conditions.ResultsSeven-day EB maturation followed by an 8-day differentiation period on OP9 cells provided the highest number of definitive (CD34+ CD235a−, 69%, p < 0.01) and lowest number of primitive (CD34− CD235a+, 1.55%, p < 0.01) precursor cells along with the highest colony-forming units (149.8 ± 11.6, p < 0.01) in feeder-free conditions. Maximal HSPC fraction (CD34+ CD38− CD45RA− CD49f+ CD90+) was 7.6–8.9% after 10 days of hematopoietic differentiation with 14.5% adult β-globin expression following RBC differentiation. Myeloid and erythroid colonies were restricted strictly to the CD34+ CD43+ fraction (370.5 ± 65.7, p < 0.001), while the CD34− CD43+ fraction produced only a small number of colonies (21.6 ± 11.9). In addition, we differentiated the CD34+ CD43+ cells towards T-lymphocytes using the OP9/DLL1 co-culture system demonstrating double-positive T cells (CD4+ CD8+) with CD3+ expression displaying a broad T cell receptor (TCR) repertoire. Confocal imaging of organoid-like structures revealed a close association of CD31+ cells with CD34+ and CD43+ cells, suggesting a potential emergence of HSPCs through endothelial to hematopoietic transition. Furthermore, fluorescently labeled organoids exhibited the emergence of spherical non-attached cells from rare progenitors at the border of the organoid center.ConclusionsIn summary, definitive HSPCs can be derived from ESCs through a dynamic cellular process from an organoid-like structure, where erythroid progeny are capable of producing adult hemoglobin and lymphoid progeny shows a diverse TCR repertoire.

Paradoxical association of TET loss of function with genome-wide DNA hypomethylation

Cancer genomes are characterized by focal increases in DNA methylation, co-occurring with widespread hypomethylation. Here, we show that TET loss of function results in a similar genomic footprint. Both 5hmC in wild-type (WT) genomes and DNA hypermethylation in TET-deficient genomes are largely confined to the active euchromatic compartment, consistent with the known functions of TET proteins in DNA demethylation and the known distribution of 5hmC at transcribed genes and active enhancers. In contrast, an unexpected DNA hypomethylation noted in multiple TET-deficient genomes is primarily observed in the heterochromatin compartment. In a mouse model of T cell lymphoma driven by TET deficiency (Tet2/3 DKO T cells), genomic analysis of malignant T cells revealed DNA hypomethylation in the heterochromatic genomic compartment, as well as reactivation of repeat elements and enrichment for single-nucleotide alterations, primarily in heterochromatic regions of the genome. Moreover, hematopoietic stem/precursor cells (HSPCs) doubly deficient for Tet2 and Dnmt3a displayed greater losses of DNA methylation than HSPCs singly deficient for Tet2 or Dnmt3a alone, potentially explaining the unexpected synergy between DNMT3A and TET2 mutations in myeloid and lymphoid malignancies. Tet1-deficient cells showed decreased localization of DNMT3A in the heterochromatin compartment compared with WT cells, pointing to a functional interaction between TET and DNMT proteins and providing a potential explanation for the hypomethylation observed in TET-deficient genomes. Our data suggest that TET loss of function may at least partially underlie the characteristic pattern of global hypomethylation coupled to regional hypermethylation observed in diverse cancer genomes, and highlight the potential contribution of heterochromatin hypomethylation to oncogenesis.

CXCL12-CXCR4 Axis Is Required for Contact-Mediated Human B Lymphoid and Plasmacytoid Dendritic Cell Differentiation but Not T Lymphoid Generation.

We investigated the involvement of CXCL12-CXCR4 interactions in human lymphohematopoiesis by coculture with telomerized human stromal cells. CXCR4 expression was low in CD34+CD38-CD45RA-CD10-CD7-CD19- immature hematopoietic stem/precursor cells (HSPCs) but higher in CD34+CD38-CD45RA+CD10+CD7+/-CD19- early lymphoid precursors and even higher in CD34+CD38+CD45RA+CD10+CD7-CD19+ pro-B cells. Inhibition of the effect of stromal cell-produced CXCL12 by an anti-CXCR4-blocking Ab suppressed the generation of CD45RA+CD10-CD7+CD19- early T lymphoid precursors (ETPs) and CD45RA+CD10+CD7-CD19+/- B lymphoid precursors on stromal cells, but it did not affect the generation of ETPs in conditioned medium of stromal cell cultures. Replating assays showed that contact with stromal cells was critical for HSPC-derived CD45RA+CD10+CD7-CD19- B lineage-biased precursors to differentiate into CD19+ pro-B cells, which was suppressed by the anti-CXCR4 Ab. Conversely, HSPC-derived ETPs possessed T and B lymphoid and monocytic differentiation potential; stromal cell contact was not required for their growth but rather promoted B lymphoid differentiation. The anti-CXCR4 Ab did not affect the growth of ETPs in conditioned medium, but it suppressed their B lymphoid differentiation on stromal cells. CD14-CD11c-HLA-DR+CD123highCD303+ plasmacytoid dendritic cells developed from HSPCs and ETPs exclusively in contact with stromal cells, which was suppressed by the anti-CXCR4 Ab. These data indicate that CXCL12 plays an essential role in stromal cell contact-mediated B lymphoid and plasmacytoid dendritic cell differentiation from immature hematopoietic and early T lymphoid precursors with a multilineage differentiation potential, but it does not participate in contact-independent generation of early T lymphoid precursors.

Competition Between (Mono)Clonal Plasma Cells and Normal Cells for Potentially Overlapping Bone Marrow Niches Is Associated with a Progressively Altered Cellular Distribution In MGUS Vs. Myeloma

AbstractAbstract 617Disappearance of normal bone marrow (BM) plasma cells (N-PC) predicts malignant transformation of monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM) into symptomatic myeloma (MM). The homing, behavior and survival of N-PC, but also CD34+ hematopoietic stem/precursor cells (HPC), B-cell precursors, and (mono)clonal/aberrant PC (M-PC) largely depends on their interaction with SDF-1 expressing BM stromal cell niches. Accordingly, it can be hypothesized that a certain degree of competition among BM B-cell precursors, CD34+ HPC, and PC exist for the same BM niches. Thus, progressive replacement of normal cells by M-PC could help to explain the occurrence of cytopenias and hypogammaglobulinemia in MM patients. However, this hypothesis has not been investigated in depth neither in MM nor in SMM and MGUS.In this study we analyze by 8-color multiparameter flow cytometry the distribution and competitive migration capacity of B-cell precursors, CD34+ HPC, N-PC and M-PC in the BM and PB of patients with MGUS (n=60), SMM (n=47) and MM (n=87) at diagnosis plus 12 MM cases studied after high-dose therapy/autologous stem cell transplantation (MM POST-HDT/ASCT) vs. healthy adults aged >60 years (HA; n=26).The percentage of BM M-PC as well as the number of M-PC from all BMPC found at diagnosis significantly (p<.001) increased from MGUS to SMM and MM patients. Circulating M-PC were also detected at diagnosis in the PB of MGUS (21%) vs. SMM (69%) and MM (75%) at increasing M-PC counts (p≤.002). Interestingly, PB vs. BM M-PC showed significantly lower amounts of sideward light scatter (p<.001), together with lower levels of CD38 (p=.001), CD40 (p=.006), CD56 (p=.03) and CD138 (p=.02) expression. Focusing on the CXCR4-SDF1 axis, we found that the proportion of CXCR4+ PC slightly decreased (p>.05) from N-PC from HA (42%) to M-PC from MGUS (39%), SMM (37%) and MM (35%) patients at diagnosis, while MM POST-HDT/ASCT cases showed the lowest values (28%). In contrast, plasma levels of SDF-1 increased from HA (1150 pg/mL) to MGUS (1352 pg/mL), SMM (1656 pg/mL) and MM (1778 pg/mL; p=.04 vs. HA) patients, returning to almost normal levels in MM POST-HDT/ASCT (1331 pg/mL). Thus, an inverse correlation trend (r2=.12; p=.05) between the proportion of CXCR4+ PC and SDF-1 plasma levels was found.Concerning the distribution of the normal cell populations, BM pro-B and pre-B cell precursors were significantly decreased in MM (p=.001) patients vs. HA, while it was normal in MGUS (p>.05) and SMM (p>.05). Despite the number of N-PC in the BM was significantly lower among MM and SMM vs. MGUS cases (p<.001), the distribution of circulating N-PC in PB was normal in all three groups of patients (p>.05 vs. HA). Interestingly however, the proportion of CXCR4+ PB N-PC progressively increased from HA (11%) to MGUS (14%), SMM (15%) and MM (21%; p=.05 vs. HA), while MM POST-HDT/ASCT cases showed the lowest median percentage of CXCR4+ PB N-PC (5%).CD34+ HPC were found to be depleted in the BM of MM (0.3%; p=.001) and SMM (0.4%; p=.002) patients vs. HA (0.9%) while MGUS (0.8%) and MM POST-HDT/ASCT (1.1%) patients showed normal CD34+ HPC numbers. Conversely, PB CD34+ HPC progressively increase from HA to MGUS, SMM and MM cases (p=.008 and p=.06 vs. HA, respectively). By contrast, in MM POST-HDT/ASCT patients the number of PB CD34+ HPC returned to normal/lower levels.Ex-vivo competition assays between BM B-cells, PC and CD34+ HPC for SDF-1 induced migration showed that in HA, CD34+ HPC displayed the highest migration potential in the presence of SDF-1, followed by pre-B cell precursors; conversely N-PC barely migrated. No significant differences were found for the migration of all cell populations analyzed between MGUS and SMM patients vs. HA, except for M-PC that showed an impaired migration in the presence of SDF-1. In turn, the migration potential of CD34+ HPC (p=.04), and pre-B cell precursors (p=.02) was markedly reduced in symptomatic MM, particularly at lower SDF-1 concentrations (30nM). Most interestingly, the migration of M-PC from symptomatic MM was markedly increased at both concentrations of SDF-1 used: median of 2.9% and 1.0% for SDF-1 concentrations of 30nM and 70nM, respectively.Overall, these findings provide evidence about the role of progressive competition and replacement of normal BM cells by M-PC in determining transformation of pre-malignant MGUS and SMM into symptomatic MM. Disclosures:No relevant conflicts of interest to declare.

Mimicking the haematopoietic niche microenvironment provides a novel strategy for expansion of haematopoietic and megakaryocyte‐progenitor cells from cord blood

Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34(+) HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT.

Transcriptional Mediator Subunit MED1/TRAP220 Acts in Stromal Cells to Support Hematopoietic Stem/Progenitor Cells through Coactivation of Osteopontin Transcription.

Abstract 250The multi-protein complex TRAP/Mediator is a subcomplex of RNA polymerase II holoenzyme. Mediator acts as the end-point integrator of a variety of activators and intracellular signaling, and conveys these signals to the general transcription machinery. Among circa 25 subunits, MED1/TRAP220 subunit is crucial for many biological events through the interaction with distinct activators such as nuclear receptors (NRs) and GATA family activators. In hematopoiesis, MED1 plays an important role for optimal NR-mediated myelomonopoiesis and GATA-1-induced erythropoiesis. In this study, we analyzed the role of MED1 in the niche. We used mouse embryonic fibroblasts (MEFs) as an in vitro niche model, since MEFs have a nuance of the osteoblastic precursor and support long-term culture-initiating cells (LTC-ICs). When syngenic normal bone marrow (BM) cells were cocultured with mitomycin C-treated Med1−/− or Med1+/+ MEFs (in p53−/− background), the number of live cells (cell counts, DNA contents and MTT assays) and DNA synthesis (BrdU incorporation) was significantly suppressed on Med1−/− MEFs compared with the control during the two-week period. However, they recovered to the control level when cocultured on Med1−/− MEFs into which MED1 was stably introduced (Rev-Med1−/− MEFs). Furthermore, when LTC-ICs were assayed in complete methylcellulose media (Methocult M3434, StemCell Technologies) after the 8-week coculture, the number of LTC-ICs was attenuated for BM cells cocultured on Med1−/− MEFs compared to the control, but recovered to the control level when cocultured on Rev-Med1−/− MEFs. In order to identify the direct target(s) of MED1 that was responsible for these phenotypes, a microarray analysis of mRNA comparing Med1+/+ and Med1−/− MEFs was performed, and disclosed approximately 15 genes whose expressions were profoundly attenuated in Med1−/− MEFs. Among molecules encoded by these genes we focused on osteopontin (OPN) because solely OPN was known to have a role in niche to support hematopoietic stem/precursor cells (HSPCs). Angiopoietin-1 and Jagged-1 expressions were comparable. The expression of Opn mRNA was distinctly downregulated in Med1−/− MEFs but recovered to the control level in Rev-Med1−/− MEFs. The Opn expression in Med1+/+ MEFs was also similar to those in MC3T3-E1 mouse osteoblastic, and OP-9 BM stromal, cells. Western blot analysis of Med1+/+ MEFs using the polyclonal antibody that recognized the N-terminus of mouse OPN disclosed the abundant expression of the full-length form of OPN (ca. 70kDa), which was much less in Med1−/− MEFs. In contrast, the N-terminal cleaved form of OPN was barely visible in both MEFs. Next a chromatin immunoprecipitation (ChIP) assay was performed to know if Mediator complex was actually recruited to the Opn promoter. As expected, when the sheared chromatin was immunopurified with the antibody against MED10 subunit, Mediator was proved to be recruited to the Opn promoter more abundantly in Med1+/+ than Med1−/− MEFs, and the recruitment in Rev−Med1−/− MEFs recovered to the level of the former. Transient transfection and luciferase reporter assays disclosed that the basal level transcription as well as vitamin D receptor (VDR)- and Runx2-mediated transcriptional activation of Opn was specifically attenuated in Med1−/− MEFs, and that the basal transcription and ligand-dependent activation were dependent on the N-terminal domain (amino acids 1 to 602) and the two NR-recognition motifs of MED1, respectively. If OPN was the direct target of MED1, OPN might be responsible for, and the addition or depletion of OPN might alter, the BM cell growth and LTC-ICs support. Indeed, the addition of recombinant full-length OPN to Med1−/− MEFs restored, and the addition of the anti-OPN (N-terminus) polyclonal antibody to Med1+/+ MEFs attenuated, both the growth of cocultured BM cells during the two-week coculture and the numbers of LTC-ICs after the long-term coculture. Finally, to exclude the possibility that these phenotypes might have been restricted to MEFs, OP-9 cells, which had widely-accepted niche function, were used for similar experiments. Indeed, the addition of the anti-OPN antibody to OP-9 cells attenuated both the growth of cocultured BM cells and the numbers of LTC-ICs. Taken together, these data suggest that MED1 in niche cells, through upregulating VDR- and Runx2-mediated transcription on the Opn promoter, plays an important role in HSPCs support. Disclosures:No relevant conflicts of interest to declare.

Genetic Network Analysis of Human CD34 + Hematopoietic Stem/Precursor Cells

Somatic CD34+ hematopoietic stem/precursor cells (HSPCs) give rise to hematopoietic cells and endothelial cells and have been used in clinical applications. Understanding the genes responsible for stemness and how they interact with each other will help us to manipulate these cells more efficiently in the future. We performed microarray analysis on human CD34+ HSPCs and on two different progeny cell types, i.e. microvascular endothelial cells and peripheral blood mononuclear cells. Systems biology and advanced bioinformatics tools were used to help clarify the genetic networks associated with these stem cell genes. We identified CD34+ HSPC genes and found that they were involved in critical biologic processes such as cell cycle regulation, chromosome organization, and DNA repair. We also identified a novel precursor gene cluster on chromosome 19p13.3. Analysis of HSPC-enriched genes using systems biology tools revealed a complex genetic network functioning in CD34+ cells, in which several genes acted as hubs to maintain the stability (such as GATA1) or connectivity (such as hepatic growth factor) of the whole network. This study provides the foundation for a more detailed understanding of CD34+ HSPCs.