Long-term Reconstitution Activity Research Articles

CD9 Marks Flt3+ Multipotent Hematopoietic Progenitors within Lsk Cells

Hematopoietic Stem Cells (HSCs) that give rise to all lineages of the blood are defined as a lineage-negative (Lin-), c-Kit+, Sca-1+(LSK) population in mouse. The expression of Flt3 on LSK cells is accompanied by loss of self-renewal capacity, and transition to lymphoid-primed multipotent progenitors (LMPPs). The magnitude of Flt3 surface abundance has been shown to be correlated with lineage potential, where megakaryocyte/erythroid (MegE) potential is incompatible with high Flt3 levels (Buza-Vidas et al. 2011). However, it is unclear whether MegE capacity is marked by Flt3 itself or restricted to a specific population within the Flt3 spectrum. Interestingly, it has been shown that commitment to MegE lineages is an early event in HSC differentiation and that MegE progenitors share several cell-surface markers with HSCs. One such marker is CD9, which is highly expressed on megakaryocyte progenitors (Nakorn, Miyamoto, and Weissman 2003)as well as on the most primitive stem cells in mouse (Karlsson et al. 2013). In fact, we have recently shown that CD9 captures all long-term reconstitution activity of the murine LSK population, even in cell fractions that lacks some of the most established HSC markers .To compare CD9- and Flt3 expression we performed FACS analysis of the LSK compartment and plotted CD9 high- and low cells on top of the CD34/Flt3 expression profile. Interestingly we found that even if CD9 expression were predominantly high in LT-(CD34-Flt3-) and ST-HSCs (CD34+Flt3-), CD9highcells reached all the way into the Flt3+population but were lost in LMPPs, as defined by the 25% highest Flt3 expressing cells. To addresswhether CD9highcells represent a distinct subpopulation within Flt3intcells, LSKCD34+Flt3int(Flt3int) and LSKCD34+FLT3intCD9high(CD9high) populations, were subjected to single-cell RNA-Sequencing analysis. Interestingly,our results demonstratethatCD9highcells are enriched in the most primitive cluster of the Flt3intpopulation. This was further validated in transplantation assays where only CD9highFlt3intcells reconstituted irradiated recipients. However, long-term reconstitution was limited to lymphoid lineages, while myeloid engraftment was only observed at early time points and self-renewal activity was absent. To address the clonal capacity of CD9highcells, we designed a protocol to assess the potential of single-cells to generate B-, Myeloid (My)- and Erythroid (Er) cells in vitro.In these conditions 68% of clones generated from CD9highcells were bipotent (55% My/B and 13% My/Er). In contrast, the majority of CD9lowcells were unipotent (32% My- and 52% B cells).Moreover,the colony forming capacity for the CD9highpopulation was significantly higher compared to CD9lowcells and when assayed for colony-forming unit-megakaryocyte (CFU-MK) capacity, Meg potential was exclusively observed in LT-HSCs, ST-HSCs as well as the CD9highFlt3intpopulation, while neither CD9lownor LMPPs generated any megakaryocyte colonies. In an attempt to improve the efficiency of MegE differentiation, single-cell clonogenic assays were performed in suspension cultures. Here,multipotency including MegE potential was gradually down regulated with increasing Flt3 cell surface expression, and decreasing CD9 expression. Together our results shows thatCD9 captures the most primitive cells within the LSKFlt3+population, including multipotent cells with MegE differentiation potential, thus resolving the heterogeneity of Flt3+hematopoietic progenitors and identifying a multipotent LSKCD9+Flt3+population distinct from LMPPs. Disclosures No relevant conflicts of interest to declare.

Rheumatoid Arthritis Causes Hematopoietic Stem Cell Reprogramming to Maintain Functionality

Rheumatoid arthritis (RA) is a debilitating autoimmune disease resulting from autoantibodies that cause damage to synovial joints. Joint damage causes increased systemic inflammatory cytokines which may lead to aberrant hematopoiesis. Indeed, RA is accompanied by many hematological complications including anemia, cytopenias, and suppressed bone marrow function. Hematopoietic stem cells (HSC) at root of the blood system can respond to inflammatory signals by activating the cell cycle and preferentially generating myeloid cells. However, chronic inflammation can also lead to HSC dysfunction. Previous studies using genetic mouse models of RA have identified myeloid overproduction in this context; however, HSC long-term reconstitution activity was maintained.To better understand hematopoietic alterations in RA, our group used the collagen induced arthritis (CIA) mouse model, which is inducible in adult mice and recapitulates many immunological features of the human disease, including elevated inflammatory cytokine levels in the bone marrow (BM) and peripheral blood (PB). Confirming prior reports, we found increased numbers of myeloid lineage cells in the PB and BM of CIA mice. We also found reduced erythroid and lymphoid lineage progenitor cell numbers in the BM, consistent with anemia and immunosenescence phenotypes in RA patients. Interestingly, these features were accompanied by a significant increase in the number of myeloid-biased multipotent progenitor-3 (MPP3) cells, suggesting increased activation of myeloid differentiation pathways. However, we found no changes to the number of activated (MPP1), short-term HSC (ST-HSC), or long-term HSC (LT-HSC) in CIA mice. Likewise, and in line with previous reports, long-term HSC potential was not reduced in CIA mice, as assessed by transplantation of either purified HSC or with unfractionated BM into irradiated recipient mice. While HSC from control and CIA donor mice displayed similar blood chimerism and lineage distribution over a 16-week period, we did observe increased proportions of donor-derived MPP3 in CIA recipient animals, further supporting an activation of myeloid HSC differentiation pathways. Overall, these results reveal underlying changes in the BM driving aberrant hematopoiesis, with the HSC pool remaining intact despite activation of a myeloid differentiation pathway.To better understand how HSC are impacted by arthritic inflammation, we assessed the molecular state of control and CIA HSC using RNA-seq. We found 292 genes significantly upregulated and 237 genes significantly downregulated in CIA HSC. Analysis of these genes using Ingenuity, GSEA, and DAVID tools revealed broad downregulation of inflammatory and proliferation signaling pathways including IL-1β, NFκB, MYC, and ERK. Genes for G1/S cell cycle transition, transcription, protein translation, and proliferation pathways were also significantly downregulated in CIA HSC. On the other hand, genes corresponding to cell cycle arrest and negative regulation of transcription were significantly upregulated. Notably, we find that IL-1β, which is produced in the BM of CIA mice, is sufficient to induce this molecular program. We find that HSC in CIA mice have a global downregulation of transcripts required for activation and proliferation, and a global upregulation of transcripts that would promote quiescence. Hence, HSC are forced back into a quiescent state by broad downregulation of cell growth and proliferation genes even during chronic inflammation caused by RA.Altogether, our data show that a mouse model of rheumatoid arthritis causes hematopoietic lineage skewing towards the myeloid lineage with simultaneous loss of lymphoid and erythroid lineage potential. Interestingly, in this chronic inflammatory setting HSC downregulate pathways involved in cytokine signaling, cell cycle activation, and translation. This mechanism can be triggered by chronic exposure to pro-inflammatory cytokines, and may serve to limit HSC proliferation and potential for damage in disease settings. These results may explain the relative rarity of outright bone marrow failure in autoimmune disease patients, while providing insight into mechanisms driving aberrant hematopoiesis in these individuals. Lastly, they provide functional evidence for cytokine blockade to normalize HSC function in the setting of RA and other chronic inflammatory diseases. DisclosuresNo relevant conflicts of interest to declare.

MiR-99 regulates normal and malignant hematopoietic stem cell self-renewal.

The microRNA-99 (miR-99) family comprises a group of broadly conserved microRNAs that are highly expressed in hematopoietic stem cells (HSCs) and acute myeloid leukemia stem cells (LSCs) compared with their differentiated progeny. Herein, we show that miR-99 regulates self-renewal in both HSCs and LSCs. miR-99 maintains HSC long-term reconstitution activity by inhibiting differentiation and cell cycle entry. Moreover, miR-99 inhibition induced LSC differentiation and depletion in an MLL-AF9-driven mouse model of AML, leading to reduction in leukemia-initiating activity and improved survival in secondary transplants. Confirming miR-99's role in established AML, miR-99 inhibition induced primary AML patient blasts to undergo differentiation. A forward genetic shRNA library screen revealed Hoxa1 as a critical mediator of miR-99 function in HSC maintenance, and this observation was independently confirmed in both HSCs and LSCs. Together, these studies demonstrate the importance of noncoding RNAs in the regulation of HSC and LSC function and identify miR-99 as a critical regulator of stem cell self-renewal.

Functional analysis of Protection of Telomeres 1a (Pot1a) in regulation of hematopoietic stem cell aging.

Repeated cell divisions induce DNA damage accumulation, which impairs stem cell function during aging. However, the general molecular mechanisms by which this occurs remain unclear. Herein, we show that the expression of protection of telomeres 1a (Pot1a), a component of shelterin, is crucial for prevention of telomeric DNA damage response (DDR) and maintenance of hematopoietic stem cell (HSC) activity during aging. We observed that HSCs express high levels of Pot1a during development, and this expression declines with aging. Knockdown of Pot1a induced an age-related phenotype, characterized by increased telomeric DDR and reduced long-term reconstitution activity. In contrast, treatment with exogenous Pot1a protein prevented telomeric DDR, which decreased stem cell activity and partially rejuvenated HSC activity. These results highlight a general, reversible mechanism by which aging compromises mammalian stem cell activity, with widespread implications for regenerative medicine.

N-cadherin+ HSCs in fetal liver exhibit higher long-term bone marrow reconstitution activity than N-cadherin− HSCs

Adult hematopoietic stem cells (HSCs) are maintained in a microenvironment known as the stem cell niche. The regulation of HSCs in fetal liver (FL) and their niche, however, remains to be elucidated. In this study, we investigated the role of N-cadherin (N-cad) in the maintenance of HSCs during FL hematopoiesis. By using anti-N-cad antibodies (Abs) produced by our laboratory, we detected high N-cad expression in embryonic day 12.5 (E12.5) mouse FL HSCs, but not in E15.5 and E18.5 FL. Immunofluorescence staining revealed that N-cad+c-Kit+ and N-cad+ endothelial protein C receptor (EPCR)+ HSCs co-localized with Lyve-1+ sinusoidal endothelial cells (ECs) in E12.5 FL and that some of these cells also expressed N-cad. However, N-cad+ HSCs were also observed to detach from the perisinusoidal niche at E15.5 and E18.5, concomitant with a down-regulation of N-cad and an up-regulation of E-cadherin (E-cad) in hepatic cells. Moreover, EPCR+ long-term (LT)-HSCs were enriched in the N-cad+Lin−Sca-1+c-Kit+ (LSK) fraction in E12.5 FL, but not in E15.5 or E18.5 FL. In a long-term reconstitution (LTR) activity assay, higher engraftment associated with N-cad+ LSK cells versus N-cad− LSK cells in E12.5 FL when transplanted into lethally irradiated recipient mice. However, the higher engraftment of N-cad+ LSK cells decreased subsequently in E15.5 and E18.5 FL. It is possible that N-cad expression conferred higher LTR activity to HSCs by facilitating interactions with the perisinusoidal niche, especially at E12.5. The down-regulation of N-cad during FL hematopoiesis may help us better understand the regulation and mobility of HSCs before migration into BM.

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

AbstractAbstract 2617Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs.On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004).In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT.These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures:No relevant conflicts of interest to declare.

Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells

AbstractThe endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM−Sca-1+ cells, osteoblast-enriched ALCAM+Sca-1−, and ALCAM–Sca-1− cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM+Sca-1− cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion–related genes in HSCs. Microarray analysis showed that ALCAM−Sca-1+ fraction highly expressed cytokine-related genes, whereas the ALCAM+Sca-1− fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic markerlow/− subpopulation in ALCAM+Sca-1− fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow.

Endothelial protein C receptor–expressing hematopoietic stem cells reside in the perisinusoidal niche in fetal liver

AbstractHematopoietic stem cells (HSCs) are maintained in specialized niches in adult bone marrow. However, niche and HSC maintenance mechanism in fetal liver (FL) still remains unclear. Here, we investigated the niche and the molecular mechanism of HSC maintenance in mouse FL using HSCs expressing endothelial protein C receptor (EPCR). The antiapoptotic effect of activated protein C (APC) on EPCR+ HSCs and the expression of protease-activated receptor 1 (Par-1) mRNA in these cells suggested the involvement of the cytoprotective APC/EPCR/Par-1 pathway in HSC maintenance. Immunohistochemistry revealed that EPCR+ cells were localized adjacent to, or integrated in, the Lyve-1+ sinusoidal network, where APC and extracellular matrix (ECM) are abundant, suggesting that HSCs in FL were maintained in the APC- and ECM-rich perisinusoidal niche. EPCR+ HSCs were in a relatively slow cycling state, consistent with their high expression levels of p57 and p18. Furthermore, the long-term reconstitution activity of EPCR+ HSCs decreased significantly after short culture but not when cocultured with feeder layer of FL-derived Lyve-1+ cells, which suggests that the maintenance of the self-renewal activity of FL HSCs largely depended on the interaction with the perisinusoidal niche. In conclusion, EPCR+ HSCs resided in the perisinusoidal niche in mouse FL.

Identification of in vitro growth conditions for c-Kit–negative hematopoietic stem cells

AbstractOur laboratory recently identified a quiescent class of pluripotent hematopoietic stem cells (PHSCs) that are lineage negative (Linneg), lack c-Kit, and are able to give rise to c-Kit–positive (c-Kitpos) PHSCs in vivo. This population fails to proliferate in vitro but has delayed reconstituting activity in vivo. In this study, we purified these cells to enrich for the PHSCs and we identified in vitro conditions capable of supporting their maturation. The c-Kit–negative (c-Kitneg) cells exhibited differential expression of Sca-1, CD34, CD43, CD45, and Thy 1.2. We purified the cells based on Sca-1, as it is expressed on active PHSCs. We detected pre–colony-forming unit spleen (pre–CFU-s) activity in both the Sca-1neg and Sca-1pos populations, indicating the presence of primitive PHSCs in both populations. However, our in vitro studies suggest that the Sca-1pos population is enriched for PHSCs. The in vitro systems that support the growth of these dormant cells include a modified long-term marrow culture and various stromal cell lines. In modified long-term bone marrow cultures, c-Kitneg cells gave rise to c-Kitpos PHSCs, with long-term reconstitution activity in vivo. Thus we have established an in vitro system to examine PHSC maturation that will allow us to study the mediators of the c-Kitneg to c-Kitpos transition.

TCDD treatment eliminates the long-term reconstitution activity of hematopoietic stem cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disrupting chemical (EDC), can cause carcinogenesis, immunosuppression, and teratogenesis, through a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Despite remarkable recent advances in stem cell biology, the influence of TCDD on hematopoietic stem cells (HSCs), which possess the ability to reconstitute long-term multilineage hematopoiesis, has not been well investigated. In this study we examined the influence of TCDD on HSCs enriched for CD34(-), c-kit(+), Sca-1(+), lineage negative (CD34-KSL) cells. The number of the CD34-KSL cells was found to be increased about four-fold upon a single oral administration of TCDD (40 micro g/kg body weight). Surprisingly, we found that these TCDD-treated cells almost lost long-term reconstitution activity. This defect was not present in AhR(-/-) mice. These findings suggest that modulation of AhR/ARNT system activity may have an effect on HSC function or survival.

Differential regulation of the human and murine CD34 genes in hematopoietic stem cells.

Human CD34 (hCD34)-positive cells are used currently as a source for hematopoietic transplantation in humans. However, in steady-state murine hematopoiesis, hematopoietic stem cells (HSCs) with long-term reconstitution activity are found almost exclusively in the murine CD34 (mCD34)-negative to low fraction. To evaluate the possible differences in hCD34 and mCD34 gene expression in hematopoiesis, we made transgenic mouse strains with human genomic P1 artificial chromosome clones spanning the entire hCD34 genomic locus. In all transgenic mouse strains, a vast majority of phenotypic and functional HSC populations including mCD34(-/lo) express the hCD34 transgene. These data strongly support the notion that hCD34(+) human bone marrow cells contain long-term HSCs that can maintain hematopoiesis throughout life.