Lymphoid Differentiation Potential Research Articles

Epigenetic reversal of hematopoietic stem cell aging in Phf6-knockout mice.

Aging is characterized by an accumulation of myeloid-biased hematopoietic stem cells (HSCs) with reduced developmental potential. Genotoxic stress and epigenetic alterations have been proposed to mediate age-related HSC loss of regenerative and self-renewal potential. However, the mechanisms underlying these changes remain largely unknown. Genetic inactivation of the plant homeodomain 6 (Phf6) gene, a nucleolar and chromatin-associated factor, antagonizes age-associated HSC decline. Immunophenotyping, single-cell transcriptomic analyses and transplantation assays demonstrated markedly decreased accumulation of immunophenotypically defined HSCs, reduced myeloid bias and increased hematopoietic reconstitution capacity with preservation of lymphoid differentiation potential in Phf6-knockout HSCs from old mice. Moreover, deletion of Phf6 in aged mice rejuvenated immunophenotypic, transcriptional and functional hallmarks of aged HSCs. Long-term HSCs from old Phf6-knockout mice showed epigenetic rewiring and transcriptional programs consistent with decreased genotoxic stress-induced HSC aging. These results identify Phf6 as an important epigenetic regulator of HSC aging.

Decline in Insulin-like Growth Factor-1 (IGF1) from Aged Mesenchymal Stromal Cells Is a Targetable Mechanism to Rescue Hematopoietic Stem Cell Aging

Hematopoietic stem cells (HSCs) are responsible for long-term maintenance and regeneration of the hematopoietic system. Loss of long-term (LT)-HSC function is a major contributor to decline in hematopoietic function with aging, leading to increased risk of infection, poor vaccination response, and increased susceptibility to hematologic malignancies. A number of LT-HSC-intrinsic alterations and LT-HSC-extrinsic changes in the bone marrow (BM) microenvironment have been associated with functional decline in aged LT-HSCs. Outstanding questions in the field are which of these mechanisms cause, rather than are a consequence of, physiological aging and what mechanism(s) represent key therapeutic targets to extend healthy LT-HSC function in older age? Previously, we took the novel approach of examining LT-HSC frequency and function in mice at a wide array of ages with the rationale that interventions to extend LT-HSC function will be most effective starting at or before the age of onset of functional hematopoietic decline. We found that canonical markers of LT-HSC aging significantly accumulate by middle age (9-12mo) in C57BL/6 mice, including increased phenotypic LT-HSC frequency, reduced regenerative capacity, myeloid lineage bias at both transcriptional and functional levels, increased gH2.AX staining, and loss of polarity of CDC42 and tubulin. Furthermore, we found by reciprocal transplantation studies of young LT-HSCs into middle-aged recipient mice and middle-aged LT-HSCs into young recipient mice that LT-HSC-extrinsic changes in the middle-aged BM microenvironment were necessary and sufficient to cause LT-HSC aging. By transcriptome analysis, we identified decreased IGF1 signaling in LT-HSCs as a candidate mechanism causing LT-HSC aging. Here, we systematically identify mesenchymal stromal cells (MSCs) as the major local producer of IGF1 in the BM of young mice and determine that this production is diminished by middle age. To evaluate the specific effect of MSC-produced IGF1 on LT-HSCs, we co-cultured LT-HSCs with Igf1conditional knockout MSCs, which was found to cause increased differentiation of LT-HSCs toward myeloid progenitor cells. In vivo, reduced IGF signaling in the BM microenvironment was modeled by transplantation of wild-type BM cells into Igf1 conditional knockout recipients, which phenocopied myeloid-biased hematopoiesis as observed in middle-aged mice. A similar myeloid-biased hematopoiesis phenotype was observed upon transplant of Igf1r conditional knockout LT-HSCs into wild-type recipients, supporting a model of direct communication between MSCs and LT-HSCs via IGF1 signaling. To determine whether restoration of IGF1 signaling had the capacity to rejuvenate middle-aged LT-HSCs, we applied short-term (24hr) in vitro treatment of recombinant IGF1. IGF1 treatment restored polarity of CDC42 and tubulin, decreased gH2.AX staining, increased in vivo regenerative capacity, and increased lymphoid differentiation potential of middle-aged LT-HSCs at the transcriptional and functional levels. Furthermore, increasing local IGF1 levels in the middle-aged BM microenvironment by transient intrafemoral injection (once per week for 4 wks) restored mature B cell frequency and decreased mature myeloid cell frequency in the BM and peripheral blood, rebalancing the composition of hematopoietic cells to that observed in young mice. At a mechanistic level, short-term IGF1 stimulation of middle-aged LT-HSCs was found to induce phosphorylation of IGF1R and the downstream signaling mediator AKT, and transcriptionally activate PI3K/AKT target genes. Open chromatin profiling by ATAC-seq revealed altered chromatin organization in middle-aged LT-HSCs in response to IGF1 stimulation, altering chromatin accessibility at promoters of genes enriched in intracellular and protein localization, regulation of transport and sequence-specific DNA binding, and enrichment of regulatory regions containing E2F2 binding sites. In summary, our data strongly support that decreased local production of IGF1 in the BM microenvironment causes many hallmarks of LT-HSC aging. We propose that restoration of local IGF1 signaling, or its downstream target pathways, represents an attractive prophylactic strategy for extending hematopoietic healthspan into older age. Disclosures Trowbridge: Fate Therapeutics: Patents & Royalties: patent license.

Hypoxia Regulates Lymphoid Development of Human Hematopoietic Progenitors

Hypoxia plays a major role in the physiology of hematopoietic and immune niches. Important clues from works in mouse have paved the way to investigate the role of low O2 levels in hematopoiesis. However, whether hypoxia impacts the initial steps of human lymphopoiesis remains unexplored. Here, we show that hypoxia regulates cellular and metabolic profiles of umbilical cord blood (UCB)-derived hematopoietic progenitor cells. Hypoxia more specifically enhances invitro lymphoid differentiation potentials of lymphoid-primed multipotent progenitors (LMPPs) and pro-T/natural killer (NK) cells and invivo B cell potential of LMPPs. In accordance, hypoxia exacerbates the lymphoid gene expression profile through hypoxia-inducible factor (HIF)-1α (for LMPPs) and HIF-2α (for pro-T/NK). Moreover, loss of HIF-1/2α expression seriously impedes NK and B cell production from LMPPs and pro-T/NK. Our study describes how hypoxia contributes to the lymphoid development of human progenitors and reveals the implication of the HIF pathway in LMPPs and pro-T/NK-cell lymphoid identities.

PF460 SINGLE CELL RNASEQ REVEALS THE IN VIVO DIFFERENTIATION POTENTIAL AND TRANSCRIPTIONAL DYNAMICS OF IMMATURE HUMAN THYMOCYTES

Background:T cell development depends on the colonization of the thymus by multipotent progenitor cells from the bone marrow. There, these precursor cells are gradually reprogrammed into committed T cell progenitors that have lost the developmental potential towards other hematopoietic lineages. While lineage tracing experiments in mice have provided evidence to which non T‐cell lineages these early T cell precursors differentiate in vivo, it remains unclear in human which blood cell lineages can originate from early T cell precursors. In vitro differentiation experiments have revealed erythroid, myeloid (DC, monocyte, granulocyte) and lymphoid (B, T, NK) differentiation potential, but whether this occurs in vivo is not clear.Aims:While lineage tracing experiments in mice have provided evidence to which non T‐cell lineages these early T cell precursors differentiate in vivo, it remains unclear in human which blood cell lineages can originate from early T cell precursors. In vitro differentiation experiments have revealed erythroid, myeloid (DC, monocyte, granulocyte) and lymphoid (B, T, NK) differentiation potential, but whether this occurs in vivo is not clear.Methods:Therefore, we have performed single cell RNA sequencing on more than 70.000 human CD34+lin‐ thymocytes that comprise the most immature thymocyte fraction of the human pediatric thymus.Results:Analysis of this dataset showed that cell cycle constitutes the majority of variation in the dataset, indicating less heterogeneity than anticipated. Indeed, using a strategy in which both clustering and trajectory inference were used, only limited branching was observed towards other, non T‐cell hematopoietic lineages, indicating a discrepancy between reported developmental potential and in vivo cell fate. Using these branching events, commitment was traced to coincide with loss of CD44 expression, confirming recent findings. In addition, we used CITEseq to correlate previously defined immature thymocyte subsets to these newly defined developmental cell states.Summary/Conclusion:In conclusion, our work suggests that the in vivo differentiation of immature human thymocytes is much more restricted compared to what in vitro differentiation experiments have suggested. In addition, we have delineated the transcriptional events that define the transition from multipotency toward T‐cell lineage commitment at the single cell level and redefine immature thymocyte subsets in human.

The nuclear receptor corepressor NCoR1 regulates hematopoiesis and leukemogenesis in vivo

AbstractEnhanced understanding of normal and malignant hematopoiesis pathways should facilitate the development of effective clinical treatment strategies for hematopoietic malignancies. Nuclear receptor corepressor 1 (NCoR1) has been implicated in transcriptional repression and embryonic organ development, but its role in hematopoiesis is yet to be fully elucidated. Here, we showed that hematopoietic-specific loss of NCoR1 leads to expansion of the hematopoietic stem cell (HSC) pool due to aberrant cell cycle entry of long-term HSCs under steady-state conditions. Moreover, NCoR1-deficient HSCs exhibited normal self-renewal capacity but severely impaired lymphoid-differentiation potential in competitive hematopoietic-reconstitution assays. Transcriptome analysis further revealed that several hematopoiesis-associated genes are regulated by NCoR1. In addition, NCoR1 deficiency in hematopoietic cells delayed the course of leukemia and promoted leukemia cell differentiation in an MLL-AF9–induced mouse model. NCoR1 and its partner, histone deacetylase 3, can modulate histone acetylation and gene transcription through binding the promoter regions of myeloid-differentiation genes. Our collective results support the critical involvement of NCoR1 in normal and malignant hematopoiesis in vivo.

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.

Septin 6 regulates engraftment and lymphoid differentiation potential of murine long-term hematopoietic stem cells

Septins are a family of filament-forming GTP-binding proteins that serve as scaffolds and diffusion barriers in various cellular processes. Septin 6 is known as a fusion partner of mixed-lineage leukemia in infant acute myeloid leukemia. The occurrence of the fusion gene is associated with a reduced expression of septin 6 itself. The role of septin 6 in hematopoiesis and whether it is involved in scaffolds within hematopoietic cells is not known. Septin 6-deficient hematopoietic stem cells (HSCs) present with an increased engraftment potential but altered lymphoid differentiation with a reduced contribution to the T-cell compartment and an increased B-cell contribution. Although multipotent progenitor cells showed a very distinct septin 6 filament organization and intracellular distribution, their function was not impaired by septin 6 deficiency. Our data therefore suggest a regulatory role for septin 6 in long-term HSC function and lymphoid lineage differentiation.

Bone Marrow CD34+Progenitor Cells from HIV-Infected Patients Show an Impaired T Cell Differentiation Potential Related to Proinflammatory Cytokines

The impact of HIV infection on the frequency and differentiation capability of CD34+ bone marrow hematopoietic progenitor cells (BM-HPCs) is still debated, having a possible primary role in antiretroviral-induced immunoreconstitution. We investigated the influence of HIV replication or proinflammatory cytokines on lymphopoietic capability of BM-HPCs from seven viremic (VR) and five nonviremic (NVR) HIV-infected patients. We found that BM-HPCs from VR patients were unable to differentiate in vitro toward T cells, and produced proinflammatory cytokines in the absence of viral replication. In contrast, the lymphoid differentiation potential of BM-HPCs was partially restored in successfully antiretroviral therapy-treated patients. We also showed that TLR8 triggering induced BM-HPCs from healthy donors to release proinflammatory cytokines affecting T cell differentiation. These data suggest that in HIV-infected patients, the lymphopoiesis capability of BM-HPCs may be modulated by a virus-driven autocrine mechanism involving proinflammatory cytokines.

Progressive alterations in multipotent hematopoietic progenitors underlie lymphoid cell loss in aging.

Declining immune function with age is associated with reduced lymphoid output of hematopoietic stem cells (HSCs). Currently, there is poor understanding of changes with age in the heterogeneous multipotent progenitor (MPP) cell compartment, which is long lived and responsible for dynamically regulating output of mature hematopoietic cells. In this study, we observe an early and progressive loss of lymphoid-primed MPP cells (LMPP/MPP4) with aging, concomitant with expansion of HSCs. Transcriptome and in vitro functional analyses at the single-cell level reveal a concurrent increase in cycling of aging LMPP/MPP4 with loss of lymphoid priming and differentiation potential. Impaired lymphoid differentiation potential of aged LMPP/MPP4 is not rescued by transplantation into a young bone marrow microenvironment, demonstrating cell-autonomous changes in the MPP compartment with aging. These results pinpoint an age and cellular compartment to focus further interrogation of the drivers of lymphoid cell loss with aging.

A novel lymphoid progenitor cell population (LSKlow) is restricted by p18INK4c

The cyclin-dependent kinase inhibitor CDKN2C (p18(INK4c)) restrains self-renewal in hematopoietic stem cells (HSCs) and participates in the development and maturation of lymphoid cells. Deficiency in p18 predisposes mice and humans to hematopoietic lymphoid malignancies such as T-cell leukemia and multiple myeloma. However, the mechanism by which p18 regulates differentiation from HSCs to lymphoid cells is poorly understood. In this study, we found that a progenitor population characterized by its expression of surface markers, Lin(-) Sca-1(+) c-Kit(low) (LSK(low)), was markedly expanded in the bone marrow of p18 knock-out (p18(-/-)) mice. This novel population possessed lymphoid differentiation potential, but not myeloid differentiation potential, both invitro and invivo. Whereas LSK(low) cells and common lymphoid progenitors (CLPs) overlapped functionally in generating lymphoid cells, they were distinct cell populations, because they had different gene expression profiles. Unlike CLPs, LSK(low) cells did not express the interleukin-7 receptor. LSK(low) cells were derived from HSCs and were independent of the p18-deleted microenvironment. This cell population may represent a previously unappreciated transitional stage from HSCs to lymphoid progenitors that is strictly restricted by p18 under physiological conditions. Likewise, LSK(low) might serve as a new cellular target of lymphoid malignances in the absence of p18.

Lymphoid lineage differentiation potential of mouse nuclear transfer embryonic stem cells

Stem cells therapy is considered as an efficient strategy for the treatment of some diseases. Nevertheless, some obstacles such as probability of rejection by the immune system limit applications of this strategy. Therefore, several efforts have been made to overcome this among which using the induced pluripotent stem cells (iPSCs) and nuclear transfer embryonic stem cell (nt-ESCs) are the most efficient strategies. The objective of this study was to evaluate the differentiation potential of the nt-ESCs to lymphoid lineage in the presence of IL-7, IL-3, FLT3-ligand and TPO growth factors in vitro. To this end, the nt-ESCs cells were prepared and treated with aforementioned growth factors for 7 and 14 days. Then, the cells were examined for expression of lymphoid markers (CD3, CD25, CD127 and CD19) by quantitative PCR (q-PCR) and flow cytometry. An increased expression of CD19 and CD25 markers was observed in the treated cells compared with the negative control samples by day 7. After 14 days, the expression level of all the tested CD markers significantly increased in the treated groups in comparison with the control. The current study reveals the potential of the nt-ESCs in differentiation to lymphoid lineage in the presence of defined growth factors.

Norepinephrine improves de novo emergence of hematopoietic cells in human pluripotent stem cell differentiation cultures

In vitro differentiation of human pluripotent stem cells (hPSCs) to hematopoietic stem cells (HSCs) could provide an unlimited source of cells for HSC transplantation for hematological disorders and malignancies. Catecholamine signaling from the peripheral nervous system has recently been implicated in the emergence of HSCs in the aorta gonad mesonephros (AGM) of mouse embryos, and our own observations of AGM and urogenital ridge explants from a 6 weeks old human embryo show neurogenic potential. We thus added norepinephrine (NE) to our hPSC differentiation system and assessed hematopoietic output. We observed 2.5-fold (n=14, 0.01) increase of cells with an HSC-like phenotype (CD43+CD34+CD38-CD90+CD45RA-) in the viable fraction compared to controls. We identified this phenotype as an early progenitor cell with lymphoid and myeloid differentiation potential as well as highest CFU content. Notably, the increase was highest in this fraction compared to more mature progenitors (CD43+CD34+cells) and total blood (CD43+), and was consistent with a 1.5-fold increase of colonies in CFU assays (n=9, p<0.01). Continuing the differentiation culture for 5 more days did not show further increase in the frequency of these early progenitors, indicating that the higher proportion of early progenitors in the presence of NE is due to increased blood emergence rather than to maintenance of these progenitors in culture. Indeed, when adding NE we observed nearly 5-fold (p= 0.05, n=14) increase in the cell fraction with the surface markers of hemogenic precursors of definitive hematopoiesis CD43-CD34highCD90high (Kennedy et al., 2012) compared to controls. As the first emerging hematopoietic cells in mouse express the β2 adrenergic receptor (Adrb2) subtype, we determined the effect of an Adrb2-specific antagonist on the NE responses. We observed that both the HSC phenotype and the CD43-CD34highCD90high fractions remained increased in the presence of the antagonist, suggesting that another adrenergic receptor subtype mediates the effect of NE in the hematopoietic emergence.

Norepinephrine Improves The Generation Of Hematopoietic Cells From Human Pluripotent Stem Cells With Increased Functional Properties

The possibility of differentiating human pluripotent stem cells (hPSCs) to hematopoietic stem cells (HSCs) could provide an unlimited source of donor cells for the treatment of hematological disorders and malignancies where HSC transplantation is required. Recently, signaling from the developing peripheral nervous system (PNS) has been implicated in the generation of HSCs in the aorta gonad mesonephros (AGM) of mouse embryos. Our own observations of AGM and urogenital ridge (UR) explants from a 6 weeks old human embryo show neurogenic potential. Since the AGM and UR are active sites of hematopoietic emergence, we hypothesize that migrating neural crest (NC) cells, precursors of the PNS, play an active role in HSC generation and maturation. Given that, at the time of HSC emergence, NC cells start expressing enzymes required for catecholamine production, we added norepinephrine (NE) to our optimized hPSC differentiation system and assessed for hematopoietic progenitor cell output. We observed approximately 50% increase of cells with an HSC immunophenotype (CD43+CD34+CD38-CD90+CD45RA-) compared to control settings (1.56 ± 0.25 fold increase, n=5 independent experiments, p=0.007). We identified this phenotype as an early progenitor cell with lymphoid and myeloid differentiation potential as well as highest colony forming unit (CFU) content. Importantly, the increase was specific to this cell fraction, since the proportion of more mature progenitors (CD43+CD34+ cells) and of total blood (CD43+) did not display a significant increase in the presence of NE (respectively, 1.053 ± 0.09 fold increase p=0.2104, and 0.97 ± 0.46 fold increase p=0.880, n=5 independent experiments). Continuing the differentiation culture for 5 additional days did not show additional increase in the frequency of these early progenitors. This indicates that higher proportion of early progenitors in the presence of NE is due to increased emergence from hemogenic endothelium rather than to maintenance of the early progenitors in culture. Improved generation of early progenitors was also consistent with an increase of colonies in the CFU assay (1.84 ± 0.29 fold increase in colony numbers n=4 independent experiments, p=0.010). The increased output of early hematopoietic progenitors was reversed when the Adrenergic Receptor β2 specific inhibitor ICI 118,551 was added together with NE, showing that the effect of NE is mediated by the activation of this receptor. Our preliminary results of transplantation in NSG mice show CD43+ human chimerism at week 6 post-transplantation 11 fold higher in mice transplanted with hematopoietic cells generated in the presence of NE compared to their counterparts transplanted with cells generated in standard conditions [2.33 ± 1.25% (n=3) vs 0.21 ± 0.02% (n=2), p=0.006]. However, since the detected human cells are predominantly of myeloid lineage (CD33+/CD15+) and the chimerism levels decrease back to levels comparable to PBS transplanted negative control at week 8-10 post-transplantation, this suggests that NE increases the homing and short term repopulation ability of pluripotent stem cell derived early hematopoietic progenitors. Disclosures:No relevant conflicts of interest to declare.

The relationship between the polymorphism of immunity genes and both aging and age-related diseases

Aging is acommon, progressive and irreversible state of multi-cell dysfunction. Immune aging mainly includes the declines of regenerative capacity and lymphoid lineage differentiation potential, the hyporesponsive to infection and vaccination, the hyperresponsive in the context of inflammatory pathology, and the increased risk of autoimmunity. The dysfunction of aged immune system accelerates the occurrence of aging and age-related diseases. The mutation of immunity genes that affect immune responses accelerates or slows aging process and age-related diseases. The frequencies of acquired immunity genes, such as immune protective HLA II DRB1*11 and DRB*16-associated haplotype, are increased in the longevity populations. The increased susceptibility of immune inflammatory response, morbidity and mortality in the elderly is often associated with decreased frequencies of anti-inflammatory factor IL-10 -1082G allele, TNF-β1 haplotype cnd10T/C, cnd25G/G, -988C/C, -800G/A, low proinflammatory fator TNFa level related extended TNF-A genotype -1031C/C, -863C/A, -857C/C, IL-6-174 CC and IFN-γ+874 T allele as well. The innate immunity genes, such as highly expressed anti-inflammatory +896 G KIR4 allele, CCR5Δ32 variant, -765 C Cox-2 allele, -1708 G and 21 C 5-Lox alleles are detected in centenarians. In age-related diseases, a higher CMV-specific IgG antibody level in elderly individuals is associated with a decreased frequency of KIR haplotypes KIR2DS5 and A1B10 and an increased frequency of MBL2 haplotypes LYPB, LYQC and HYPD that result in the absence of MBL2 protein. The increased frequencies of CRP ATG haplotypes and CFH 402 His allele indicate high mortality in the elderly. In the present study, we review the advances in the polymorphism and haplotype of innate and adoptive immunity genes, and their association with both aging and age-related diseases. To strengthen the analysis of extended haplotypes, epigenetic studies of immunity genes and genetic study of hematopoietic stem cell senescence will be helpful to understand the accurate basis of aging-related immune genetics better.

Pbx1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors

The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways, including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains the lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.

Age-Associated Changes in the Differentiation Potentials of Human Circulating Hematopoietic Progenitors to T- or NK-Lineage Cells

Age-associated changes of T and NK cell (T/NK) potential of human hematopoietic stem cells are unknown. In this study, we enumerate and characterize T/NK precursors among CD34(+)Lin(-) cell populations circulating in normal human adult peripheral blood (PB) by a limiting-dilution assay using coculture with OP9-DL1 stroma cells expressing Notch 1 ligand, Delta-like 1. The frequency of T cell precursors in CD34(+)Lin(-) cells was found to decrease with donor age, whereas the ratio of NK to T cell precursor frequency (NK/T ratio) increased with age, suggesting that lymphoid differentiation potential of PB progenitors shifts from T to NK cell lineage with aging. Clonal analyses of CD34(+)Lin(-) cells showed that differences in the NK/T ratio were attributable to different distributions of single- and dual-lineage T/NK precursor clones. Because nearly all of the clones retained monocyte and/or granulocyte differentiation potentials in coculture with OP9-DL1 cells, T/NK precursors in PB are considered to be contained in the pool of T/NK/myeloid multipotent progenitors. The age-associated increase in NK over T cell commitment might occur in precursor cells with T/NK/myeloid potential.

Lymphoid Progenitor Cells from Childhood Acute Lymphoblastic Leukemia Are Functionally Deficient and Express High Levels of the Transcriptional Repressor Gfi-1

Acute lymphoblastic leukemia (ALL) is the most frequent malignancy of childhood. Substantial progress on understanding the cell hierarchy within ALL bone marrow (BM) has been recorded in the last few years, suggesting that both primitive cell fractions and committed lymphoid blasts with immature stem cell-like properties contain leukemia-initiating cells. Nevertheless, the biology of the early progenitors that initiate the lymphoid program remains elusive. The aim of the present study was to investigate the ability of lymphoid progenitors from B-cell precursor ALL BM to proliferate and undergo multilineage differentiation. By phenotype analyses, in vitro proliferation assays, and controlled culture systems, the lymphoid differentiation potentials were evaluated in BM primitive populations from B-cell precursor ALL pediatric patients. When compared to their normal counterparts, functional stem and progenitor cell contents were substantially reduced in ALL BM. Moreover, neither B nor NK or dendritic lymphoid-cell populations developed recurrently from highly purified ALL-lymphoid progenitors, and their proliferation and cell cycle status revealed limited proliferative capacity. Interestingly, a number of quiescence-associated transcription factors were elevated, including the transcriptional repressor Gfi-1, which was highly expressed in primitive CD34+ cells. Together, our findings reveal major functional defects in the primitive hematopoietic component of ALL BM. A possible contribution of high levels of Gfi-1 expression in the regulation of the stem/progenitor cell biology is suggested.

Pbx1 restrains myeloid maturation while preserving lymphoid potential in hematopoietic progenitors

The capacity of the hematopoietic system to promptly respond to peripheral demands relies on adequate pools of progenitors able to transiently proliferate and differentiate in a regulated manner. However, little is known about factors that may restrain progenitor maturation to maintain their reservoirs. Conditional knockout mice for the Pbx1 proto-oncogene have a significant reduction in lineage-restricted progenitors in addition to a profound defect in hematopoietic stem cell (HSC) self-renewal. Through analysis of purified progenitor proliferation, differentiation capacity and transcriptional profiling, we demonstrate that Pbx1 regulates the lineage-specific output of multipotent and oligopotent progenitors. In the absence of Pbx1 multipotent progenitor (MPP) and common myeloid progenitor (CMP) pools are reduced due to aberrantly rapid myeloid maturation. This is associated with premature expression of myeloid differentiation genes and decreased maintenance of proto-oncogene transcriptional pathways, including reduced expression of Meis1, a Pbx1 dimerization partner, and its subordinate transcriptional program. Conversely, Pbx1 maintains the lymphoid differentiation potential of lymphoid-primed MPPs (LMPPs) and common lymphoid progenitors (CLPs), whose reduction in the absence of Pbx1 is associated with a defect in lymphoid priming that is also present in CMPs, which persistently express lymphoid and HSC genes underlying a previously unappreciated lineage promiscuity that is maintained by Pbx1. These results demonstrate a role for Pbx1 in restraining myeloid maturation while maintaining lymphoid potential to appropriately regulate progenitor reservoirs.

Clonal heterogeneity in patients with cytogenetically normal acute myeloid leukemia with NPM1 mutations

The nucleophosmin 1 (NPM1) gene is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 60% of adult cytogenetically normal AML (CN-AML). To date, these mutations have only been detected in cells of the myeloid lineage, whereas the potential clonal involvement of the lymphoid lineage is controversial. In our study, NPM1 mutations were analyzed using the highly sensitive real-time quantitative polymerase chain reaction (RQ-PCR) method on fluorescence-activated cell-sorted (FACS) purified different circulating mature cell populations in patients with NPM1-mutated CN-AML. As expected, NPM1 mutations were found in myeloid blood cells, including CD14+ monocytes and CD66b+ granulocytes. However, we were also able to detect NPM1 mutations in CD19+ B cells and CD3−14−16+56+ natural killer (NK) cells, albeit at lower levels. Surprisingly, mutations were also detected in CD3+ T cells from all analyzed patients. Our data demonstrate that NPM1-mutated CN-AML originates in an early stem cell with both lymphoid and myeloid differentiation potential.

Stable Long-Term Blood Formation by Stem Cells in Murine Steady-State Hematopoiesis

Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis.