Subpopulations Of Hematopoietic Progenitor Cells Research Articles

Human Embryonic Stem Cells as a Model for Hematopoietic Stem Cell Differentiation and Viral Infection.

Pluripotent human embryonic stem cell (hESC) lines are a valuable in vitro tool to differentiate specific cell lineages, including cells from all three germ layers, i.e., neuronal cells, myocytes, and hematopoietic cells, including progenitors (described here), lymphoid cells, and myeloid cells. However, dramatically different cell subtypes and functional properties of specific cells can arise depending on the differentiation technique used. We previously optimized hematopoietic stem cell differentiation from two different NIH-approved hESC lines to generate CD34+ hematopoietic progenitor cells (HPCs). Infection of these HPCs with a common herpesvirus (human cytomegalovirus) results in maintenance of viral latency, capability of viral reactivation, recapitulation of viral mutant phenotypes, and virus-induced myelosuppression of hematopoietic differentiation. However, different HPC subpopulations support different viral latency and reactivation phenotypes, and different hESC-to-HPC differentiation methods alter the ratio of stem cell subsets. In addition, differences in differentiation methods are dependent on both protocol/reagents and user techniques. Here, we report a simplified and optimized method to generate large numbers of CD34+ HPCs with consistent phenotypes and demonstrate a comparison of several common methods that can be used to control the ratio of available HPC subpopulations. A key aspect of this approach is that we achieve consistency in differentiation across users in different laboratories and, importantly, among newly trained individuals. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance of human embryonic stem cells (hESCs) Basic Protocol 2: Differentiation of hESCs to hematopoietic progenitor cells (HPCs) Basic Protocol 3: Downstream functional differentiation of hESC-derived HPCs to mature lineages Support Protocol 1: Freezing and testing frozen batches of hESCs Support Protocol 2: Counting hESCs Support Protocol 3: Phenotyping by flow cytometry.

Identification of Candidate Progenitor Populations Which Causes Transient Abnormal Myelopoiesis

Transient abnormal myelopoiesis (TAM) is a temporal preleukemic status specific to neonates of Down syndrome (DS). TAM is characterized by leucocytosis and/or thrombocytopenia with the trisomy of chromosome 21 and somatic GATA1 mutation. Morphologically, most blasts found in TAM are myeloblasts, and some other types of blasts are similar to those typically found in DS-related acute megakaryoblastic leukemia (DS-AMKL). Although TAM patients usually experience spontaneous remission within 6 months, 20 to 30 % of develop DS-AMKL. The cell population that causes TAM phenotypes in vitro or in vivo mouse and human models has not yet been determined.Recently, the hematopoietic differentiation systems from human pluripotent stem cells (PSCs) have revealed the pathogenesis in various diseases including TAM. Previous TAM models have succeeded in recapitulating the phenotypes, such as arrest and/or skewness of differentiation from immature hematopoietic progenitor cells (HPCs). However, identification of the responsible cell population for TAM at a precise subpopulation level based on functional evaluations has not yet been accomplished. Therefore, we applied our 2D-hematopoietic differentiation system for identifying the responsible cell population through the progenitor assay. A comprehensive understanding of TAM pathogenesis will enable us to predict and eventually prevent the disease progression from TAM into life-threatening DS-AMKL.First, we differentiated artificial trisomy 21 embryonic stem cells (Ts21-ESCs) generated by transfer of human artificial chromosome (HAC) 21 in our 2D-differentiation system. Five subpopulations of HPCs were harvested at day6 and day9, and cells were subjected to expression analysis of hematopoiesis-related genes. Principal component analysis (PCA) successfully mapped each HPC subpopulation corresponding to the hematopoietic developmental pathways, proving that our system can be used for isolating functional HPC subpopulations.Next, we used genome edition to establish strictly controlled isogenic ES/induced pluripotent stem cell (iPSC) pairs derived from TAM, non-TAM/DS patients and the Ts21-ESCs. Then we differentiated ES/iPSCs into each hematopoietic lineage to identify the abnormal phenotypes in vitro . GATA1 -mutated clones (G1s-clones) showed obviously different phenotypes, compared to GATA1-WT clones (WT-clones). In particular, completely restricted erythroid lineage cells (CD42b-CD71+CD235a+) and increased myeloid lineage cells (CD34-CD235a-CD41-CD42b-CD43+CD45+) were observed in G1s-clones. Moreover, in megakaryocytic lineage, the frequency of immature (CD235a-CD41+CD42bdim) and mature (CD235a-CD41+CD42bbright) cells differed between G1s- and WT-clones. Notably, the proportion of immature megakaryoblasts increased in G1s-clones. These data indicated that we have succeeded in recapitulation the TAM phenotype with our in vitro differentiation system.Then, we performed progenitor assay based on in vitro TAM phenotypes. We differentiated 5 isolated HPCs subpopulation into erythrocyte, megakaryocyte and myeloid cells with a suspension culture and colony forming assay. As a result, we found strong correlations between the increased or decreased specific cell fractions and abnormal phenotypes. Correlation pattern is slightly different between clones.In conclusion, we have identified putative responsible cell populations that contribute to the pathological phenotype in TAM. These populations were located on a series of spectra in the differentiation stage. Ubiquitous expression of GATA1 during the differentiation process and wide heterogeneity of the differentiation systems in general may be the reason why precise identification of the single responsible population was not possible. However, our model of TAM using iPSCs enabled us to narrow down the candidate cell populations which have the potential to be a source of DS-AMKL. The identification of responsible progenitor cells is an important first step to predict the transformation from TAM into DS-AMKL and to prevent its progression. Moreover, the method used in this study, such as 2D-differentiation system and progenitor assay, is a promising way to identify the responsible cell population of various types of leukemia other than TAM. DisclosuresNo relevant conflicts of interest to declare.

Fate Decision Between Group 3 Innate Lymphoid and Conventional NK Cell Lineages by Notch Signaling in Human Circulating Hematopoietic Progenitors.

The role of Notch signaling in human innate lymphoid cell (ILC) differentiation is unclear, although IL-7 and IL-15 promote differentiation of natural cytotoxicity receptor (NCR) NKp44+ group 3 ILCs (NCR+ILC3s) and conventional NK (cNK) cells from CD34+ hematopoietic progenitor cells (HPCs) ex vivo. In this study, we analyzed the functions of Notch in the differentiation of NCR+ILC3s and cNK cells from human HPC subpopulations circulating in peripheral blood by limiting dilution and clonal assays using high-throughput flow cytometry. We demonstrated that Notch signaling in combination with IL-7 induced NCR+ILC3 differentiation, but conversely suppressed IL-15-dependent cNK cell generation in CD45RA+Flt-3-c-Kitlow, a novel innate lymphocyte-committed HPC subpopulation. In contrast, Notch signaling induced CD45RA-Flt-3+c-Kithigh multipotent HPCs to generate CD34+CD7+CD62Lhigh, the earliest thymic progenitor-like cells, which preserved high cNK/T cell potential, but lost NCR+ILC3 potential. These findings implicate the countervailing functions of Notch signaling in the fate decision between NCR+ILC3 and cNK cell lineages at different maturational stages of human HPCs. Inhibition of Notch functions by Abs specific for either the Notch1 or Notch2 negative regulatory region suggested that both Notch1 and Notch2 signals were involved in the fate decision of innate lymphocyte-committed HPCs and in the generation of earliest thymic progenitor-like cells from multipotent HPCs. Furthermore, the synergistic interaction between Notch and IL-7 in NCR+ILC3 commitment was primarily explicable by the induction of IL-7 receptor expression in the innate lymphocyte-committed HPCs by Notch stimulation, suggesting the pivotal role of Notch in the transcriptional control required for human NCR+ILC3 commitment.

Population Balance Reconstruction of the Hematopoietic Differentiation Hierarchy

Fate commitment of hematopoietic progenitor cells (HPCs) is thought to occur through a series of hierarchical fate choices, established over the past four decades through live cell tracking, in vitro colony-forming assays and transplantation of defined sub-populations of HPCs. Depictions of the HPC hierarchy invoke a tree structure, with gradual lineage-restriction at branch points, although the precise tree remains controversial. A second controversy relates to the nature of undifferentiated MPPs. MPPs have been suggested to express conflicting lineage-restricted programs indicating multi-lineage priming, or to host distinct cell sub-populations with intrinsic biases, or alternatively to form an entirely na•ve state. We asked whether the molecular state of cells seen by RNA profiling of thousands of single HPCs could resolve these controversies, by correctly predicting the known fates of sub-populations of HPCs reported over the past two decades, defining MPP heterogeneity, and defining the topology of HPC fate commitment.Predicting the future behavior of cells from high-dimensional snapshots of their current state is an unsolved problem. Until now, snapshots of single cell molecular states have been used to order events in cell differentiation, cell cycle, and perturbation response by methods that fit cell states to a curve or a tree, but these approaches tend to be suggestive rather than predictive. Here, we invoke a conservation law Ð commonly known as "population balance" Ð to formally predict differentiation fates from single cell molecular profiles, using an approach that is asymptotically exact given certain assumptions about the differentiation process. Application of the conservation law to single cell RNA profiles required developing novel mathematical results, leading to an algorithm termed Population Balance Analysis (PBA).We apply PBA to bone marrow hematopoiesis, and recover the structure of the hematopoietic progenitor cell (HPC) population as it differentiates into seven lineages. The inferred fate choices reconcile fate-potential assays from the past two decades, and suggest a unified molecular definition of the hematopoietic hierarchy. In contrast to the canonical hierarchy, we predict that HPCs do not form a strict tree, and that MPPs show evidence of multi-lineage priming with simultaneous low-level gene expression of conflicting differentiation programs. In normal hematopoiesis, we predict a novel erythroid-basophil progenitor cell state, whereas erythroid/megakaryocyte fates become coupled in stress. We predict several novel regulators of cell fate at choice points in hematopoiesis. Overall, this work reconciles novel RNA-Seqdata with gold standard functional fate assays; it predicts novel regulators of hematopoiesis; and it establishes a predictive analytical method for dissecting complex differentiation hierarchies from single cell molecular profiles. [Display omitted] DisclosuresKlein:OneCell Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells.

Derivation of hematopoietic stem cells (HSCs) from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom(+) hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of the tdTom(+) population. Notably, RUNX1c/tdTom(+) cells represent only a limited subpopulation of the CD34(+) CD45(+) and CD34(+) CD43(+) cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom(+) cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom(+) population to CD34(+) CD45(+) umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs.

RUNX1c Expression Identifies an Enriched Subpopulation of Hematopoietic Progenitor Cells Derived From Human Pluripotent Stem Cells.

Abstract 2294The ability of human embryonic stem cells (hESCs) to differentiate into any cell lineage makes them an important resource for studies of developmental biology. hESC-derived hematopoietic stem cells (HSCs) developed through in vitro culture systems could provide an unlimited supply of material for replacement of defective blood lineages in vivo. However, to date, there has been only limited ability to isolate functional HSCs from hESCs. Runx1 is a key hematopoietic transcription factor vital to the development of the early embryo. Previous studies have demonstrated the onset of Runx1c to correlate directly with the emergence of definitive HSCs in the murine embryo. Similarly, expression of RUNX1c increases during differentiation of hESCs to hematopoietic cells. This particular feature makes RUNX1c an especially attractive tool for tracking the development of potential HSCs from hESCs. Therefore, we have created a novel reporter system in hESCs wherein both 1 kb of the basal promoter as well as the +24 intronic enhancer of RUNX1c drive expression of the tdTomato fluorochrome. Transgenic hESC lines stably expressing tdTomato from RUNX1c regulatory elements were generated using the Sleeping Beauty transposon system. Successfully engineered clones were selected based on constitutive expression of a GFPzeocin fusion protein which was also included in the transgene. To induce differentiation, the spin-embryoid body (EB) method was used with fully defined media. Differentiating cells were analyzed for tdTomato expression. Sorted tdTomato+ cells were evaluated for RUNX1c expression and hematopoietic stem/progenitor cell potential (CFC assay). Undifferentiated RUNX1c reporter hESC clones were uniformly tdTomato-negative. These clones are shown to express the tdTomato transgene concurrent with the expression of endogenous RUNX1c, as analyzed by flow cytometry and qRT-PCR. Expression was first seen at day 12 with a peak between days 14–16 of differentiation. Cells sorted for expression of tdTomato showed a significant increase of endogenous RUNX1c expression compared to tdTomato-negative cells. Using fluorescence microscopy, we were able to detect tdTomato+ cells with hematopoietic morphology budding from the EBs after 12–15 days. Together, these results validate the accuracy of this reporter system. tdTomato+ cells were found to be enriched for hematopoietic progenitor potential, as demonstrated by increased ability to form hematopoietic colonies in methylcelluose (430 +/− 143 per 105 cells) as compared to both tdTomato negative (131 +/− 15 per 105 cells) and unsorted cells (163 +/− 28 per 105 cells). Interestingly and unexpectedly, flow cytometric analysis demonstrated that only 5–10% of CD34+CD45+ cells expressed tdTomato. The first CD45+ population was found to lack tdTomato expression. Likewise, the first emerging tdTomato+ cells did not express CD45. Of the extracellular markers examined in this study, CD31 and CD43 were found to be expressed on all tdTomato+ cells, while CD31+ and CD43+ populations, though initially tdTomato−, were found to gradually increase in tdTomato expression to 80% and 90% by day 20, respectively. Emerging CD34+ cells lack tdTomato expression with an increase to around 10% of cells, plateauing at day 15. While CD45+ cells initially lack tdTomato expression, they gain expression over time, until days 19–22, when 50–90% of CD45+ cells co-express tdTomato (RUNX1c). Therefore, this hESC RUNX1c reporter system appears to accurately define timing of RUNX1c expression. The finding that emerging RUNX1c+ cells have increased hematopoietic progenitor cell potential mimics what has been found in mice. This, combined with our finding that only a fraction of CD34+CD45+ cells express Runx1c, suggests that one reason hESC-derived hematopoietic cells fail to engraft in immunodeficient mice is that only a small percent of differentiated cells that express hematopoietic surface antigens actually have hematopoietic stem/progenitor cell activity. Current studies focus on more in-depth analysis of tdTomato expressing cells using both single-cell qRT-PCR as well as LTC-IC and engraftment assays to test for functionality. Furthermore, we are currently testing an iPSC line stably expressing the same RUNX1c:tdTomato transgene to test how its RUNX1c expression in iPSCs compares to hESCs during hematopoietic differentiation. Disclosures:No relevant conflicts of interest to declare.

The early phase of engraftment after murine blood cell transplantation is mediated by hematopoietic stem cells.

Blood cells transplantation is largely replacing bone marrow transplantation because engraftment is more rapid. This accelerated engraftment is thought to be mediated by relatively mature committed hematopoietic progenitor cells. Herein, we have used a modified rhodamine (Rho) staining procedure to identify and purify Rho+/++ (dull/bright) and Rho- (negative) subpopulations of hematopoietic progenitor cells in murine cytokine-mobilized blood. The Rho+/++ cell population contained > 99% of committed progenitor cells with in vitro colony-forming ability. The Rho- cell population contained the majority of hematopoietic stem cells with in vivo marrow repopulating ability. The rate of hematopoietic reconstitution was identical in recipients of grafts containing only purified Rho- stem cells or purified Rho- stem cells in combination with large numbers of Rho+/++ committed progenitor cells. In contrast, transplantation of 3-fold more hematopoietic stem cells resulted in accelerated reconstitution, indicating that the reconstitution rate was determined by the absolute numbers of Rho- stem cells in the graft. In addition, we observed a 5- to 8-fold reduced frequency of the subset of hematopoietic stem cells with long-term repopulating ability in cytokine-mobilized blood in comparison to steady-state bone marrow. Our results indicate that hematopoietic stem cells and not committed progenitor cells mediate early hematopoietic reconstitution after blood cell transplantation and that relative to bone marrow, the frequency of stem cells with long-term repopulating ability is reduced in mobilized blood.

Detection of trisomy 12 in CD34+ progenitor cells in a patient with B-cell chronic lymphocytic leukemia by florescence in situ hybridization

B-cell chronic lymphocytic leukemia (B-CLL) is a slowly progressive disease resulting from the clonal expansion of mature B lymphocytes. The most frequent chromosomal abnormality is trisomy 12. Recently more aggressive therapeutic approaches using myeloablative therapy and autologous stem-cell support have been developed. Phase I/II studies have resulted in molecular remission and prolonged survival. One cause of relapse may be tumor-cell contamination of the transplant. We asked whether immunophenotypically identified hematopoietic progenitor cells are part of the malignant cell population in B-CLL. In a patient with trisomy 12, two subpopulations of hematopoietic progenitor cells, CD34+/CD38+ and CD34+/CD38- cells, were isolated by fluorescence-activated-cell-sorting; the sort purity was 98%. Trisomy 12 was detected in 13% of CD34+/38+ cells and in 34% of CD34+/38- cells. These data suggest that CD34+ cells are involved in the malignant clone of patients with B-CLL. The results are of significance for future strategies using autologous stem-cell transfusion.

Chemical suppression of a subpopulation of primitive hematopoietic progenitor cells: 1,3-butadiene produces a hematopoietic defect similar to steel or white spotted mutations in mice.

Chronic exposure of mice to 1,3-butadiene produces a macrocytic-megaloblastic anemia, thymic hypoplasia, and an increased incidence of T-cell lymphoma/leukemia. This is reminiscent of pathologies observed in mice bearing mutations at the W and Sl loci, which are deficient in c-kit and c-kit ligand (CKL), respectively. The influence of 3,4-epoxybutene (EB), the primary metabolite of 1,3-butadiene, on the colony-forming response of hematopoietic progenitor cells (HPCs) from C57BL/6, Sl, and W mice was investigated in order to elucidate the role of altered HPC regulation in the pathogenesis of 1,3-butadiene toxicity. EB pretreatment suppressed interleukin 3 colony formation and abrogated CKL synergism of the granulocyte-macrophage/colony-stimulating factor (GM-CSF) response in C57BL/6 cells, had no effect on colony formation induced by GM-CSF or granulocyte/colony-stimulating factor (G-CSF) alone, and failed to suppress CKL-induced synergism of the G-CSF response. Experiments conducted with cells from Sl and W mice revealed that they lack the same primitive HPC targeted by EB. EB pretreatment in vitro and butadiene exposure in vivo mimic hematopoietic defects seen in W and Sl mice, suggesting that the pleotypic pathologies encountered in these murine models may be largely due to a common defect in primitive HPCs. Susceptibility to EB appears to define a functional subpopulation of primitive HPCs and illustrates that differences observed in the susceptibility of specific cytokine responses to chemical/drug exposure may provide a valuable tool for characterizing functional subpopulations of HPCs.