scholarly journals Neogenin-1 distinguishes between myeloid-biased and balancedHoxb5+mouse long-term hematopoietic stem cells

2019 ◽  
Author(s):  
Gunsagar S. Gulati ◽  
Monika Zukowska ◽  
Joseph Noh ◽  
Allison Zhang ◽  
Rahul Sinha ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Surface Marker ◽  
Cycle Phase ◽  
Hematopoietic Stem ◽  
Prospective Isolation

ABSTRACTHematopoietic stem cells (HSCs) self-renew and generate all blood cells. Recent studies with single-cell transplants (1–3) and lineage tracing (4, 5) suggest that adult HSCs are diverse in their reconstitution and lineage potentials. However, prospective isolation of these subpopulations has remained challenging. Here, we identify Neogenin-1 (NEO1) as a unique surface marker on a fraction of mouse HSCs labeled withHoxb5, a specific reporter of long-term HSCs (LT-HSCs) (6). We show that NEO1+Hoxb5+LT-HSCs expand with age and respond to myeloablative stress, while NEO1−Hoxb5+LT-HSCs exhibit no significant change in number. NEO1+Hoxb5+LT-HSCs are more often in the G2/S cell cycle phase compared to NEO1−Hoxb5+LT-HSCs in both young and old bone marrow. Upon serial transplantation, NEO1+Hoxb5+LT-HSCs exhibit myeloid-biased differentiation and reduced reconstitution, while NEO1−Hoxb5+LT-HSCs are lineage-balanced and stably reconstitute recipients. Gene expression comparison reveals increased expression of cell cycle genes and evidence of lineage-priming in the NEO1+fraction. Finally, transplanted NEO1+Hoxb5+LT-HSCs rarely generate NEO1−Hoxb5+LT-HSCs, while NEO1−Hoxb5+LT-HSCs repopulate both LT-HSC fractions. This supports a model in which dormant, balanced, NEO1−Hoxb5+LT-HSCs can hierarchically precede active, myeloid-biased NEO1+Hoxb5+LT-HSCs.SIGNIFICANCE STATEMENTHematopoietic stem cells (HSCs) are rare cells that have the unique ability to regenerate themselves and produce all blood cells throughout life. However, HSCs are functionally heterogeneous and several studies have shown that HSCs can differ in their contribution to major blood lineages. In this study, we discovered that the surface marker, Neogenin-1, can divide mouse HSCs into two subpopulations—one that is more active but biased towards producing myeloid cells and another that is more dormant and capable of equally producing all blood lineages. Neogenin-1 reveals the diversity and hierarchical relationship of HSCs in the mouse bone marrow, enables the prospective isolation of myeloid-biased and balanced HSCs, and opens opportunities to do the same in humans.

Mesenchymal Stem Cells from the Wharton’s Jelly of Umbilical Cord Segments Support the Maintenance of Long Term Culture-Initiating Cells from Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3650-3650
Author(s):  
Kent W. Christopherson ◽  
Tiki Bakhshi ◽  
Shamanique Bodie ◽  
Shannon Kidd ◽  
Ryan Zabriskie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Blood Cells ◽  
Hematopoietic Stem ◽  
Cord Blood Cells

Abstract Hematopoietic Stem Cells (HSC) are routinely obtained from bone marrow, mobilized peripheral blood, and umbilical Cord Blood. Traditionally, adult bone marrow has been utilized as a source of Mesenchymal Stem Cells (MSC). Bone marrow derived MSC (BM-MSC) have previously been shown to maintain the growth of HSC obtained from cord blood and have been utilized for cord blood expansion purposes. However, the use of a mismatched BM-MSC feeder stromal layer to support the long term culture of cord blood HSC is not ideal for transplant purposes. The isolation of MSC from a novel source, the Wharton’s Jelly of Umbilical Cord segments, was recently reported (Romanov Y, et al. Stem Cells.2003; 21: 105–110) (Lee O, et al. Blood.2004; 103: 1669–1675). We therefore hypothesized that Umbilical Cord derived MSC (UC-MSC) have the ability to support the long term growth of cord blood derived HSC similar to that previously reported for BM-MSC. To test this hypothesis, MSC were isolated from the Wharton’s Jelly of Umbilical Cord segments and defined morphologically and by cell surface markers. UC-MSC were then tested for their ability to support the growth of pooled CD34+ cord blood cells in long term culture - initiating cell (LTC-IC) assays as compared to BM-MSC. We observed that like BM-MSC, CB-MSC express a defined set of cell surface markers. By flow cytometry we determined that that both UC-MSC and BM-MSC are positive for CD29, CD44, CD73, CD90, CD105, CD166, HLA-A and negative for CD45, CD34, CD38, CD117, HLA-DR expression. Utilizing Mitomycin C treated (200 μM, 15 min.) UC-MSC from multiple donors as a feeder layer we observed that UC-MSC have the ability to support the maintenance of long term hematopoiesis during the LTC-IC assay. Specifically, UC-MSC isolated from separate umbilical cord donors support the growth of 69.6±11.9 (1A), 31.7±3.9 (2B), 67.0±13.5 (3A), and 38.5±13.7 (3B) colony forming cells (CFC) per 1×104 CD34+ cord blood cells as compared to 64.0±4.2 CFC per 1×104 CD34+ cord blood cells supported by BM-MSC (Mean±SEM, N=4 separate segments from three different donors). Thus, Umbilical Cord derived Mesenchymal Stem Cells, a recently described novel source of MSC, have the ability to support long term maintenance of Hematopoietic Stem Cells, as defined by the LTC-IC assay. These results may have potential therapeutic application with respect to ex vivo stem cell expansion of Cord Blood Hematopoietic Stem Cells utilizing a Mesenchymal Stem Cell stromal layer. In addition, these data suggest the possibility of co-transplantation of matched Mesenchymal and Hematopoietic Stem Cells from the same umbilical cord and cord blood donor respectively. Lastly, these results describe a novel model system for the future study of the interaction between Cord Blood Hematopoietic Stem Cells and the appropriate supportive microenvironment represented by the Umbilical Cord - Mesenchymal Stem Cells.

The Endothelial Antigen ESAM Marks Hematopoietic Stem Cells throughout Life

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 727-727 ◽  
Author(s):  
Takafumi Yokota ◽  
Kenji Oritani ◽  
Stefan Butz ◽  
Koichi Kokame ◽  
Paul W Kincade ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Endothelial Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Fetal Liver ◽  
Side Population ◽  
Expression Profiles ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSC) are an important cell type with the capacity for self-renewal as well as differentiation into multi-lineage blood cells, maintaining the immune system throughout life. Many studies have attempted to identify unique markers associated with these extremely rare cells. In bone marrow of adult mice, the Lin-c-kitHi Sca1+ CD34−/Lo Thy1.1Lo subset is known to include HSC with long-term repopulating capacity. However, several of these parameters differ between strains of mice, change dramatically during developmental age and/or are expressed on many non-HSC during inflammation. Efficient HSC-based therapies and the emerging field of regenerative medicine will benefit from learning more about what defines stem cells. We previously determined that the most primitive cells with lymphopoietic potential first develop in the paraaortic splanchnopleura/aorta-gonad-mesonephros (AGM) region of embryos using Rag1/GFP knock-in mice. We also reported that Rag1/GFP-c-kitHi Sca1+ cells derived from E14.5 fetal liver (FL) reconstituted lympho-hematopoiesis in lethally irradiated adults, while Rag1/GFPLo c-kitHi Sca1+ cells transiently contributed to T and B lymphopoiesis. To extend those findings, microarray analyses were conducted to search for genes that characterize the initial transition of fetal HSC to primitive lymphopoietic cells. The comparisons involved mRNA from Rag1Lo ckitHi Sca1+, early lymphoid progenitors (ELP) and the HSC-enriched Rag1-ckitHi Sca1+ fraction isolated from E14.5 FL. While genes potentially related to early lymphopoiesis were discovered, our screen also identified genes whose expression seemed to correlate with HSC. Among those, endothelial cell-selective adhesion molecule (ESAM) attracted attention because of its conspicuous expression in the HSC fraction and sharp down-regulation on differentiation to ELP. ESAM was originally identified as an endothelial cell-specific protein, but expression on megakaryocytes and platelets was also reported (J. Biol. Chem., 2001, 2002). Flow cytometry analyses with anti-ESAM antibodies showed that the HSC-enriched Rag1-c-kitHi Sca1+ fraction could be subdivided into two on the basis of ESAM levels. The subpopulation with the high density of ESAM was enriched for c-kitHi Sca1Hi cells, while ones with negative or low levels of ESAM were found in the c-kitHi Sca1Lo subset. Among endothelial-related antigens on HSC, CD34 and CD31/PECAM1 were uniformly present on Rag1-c-kitHi Sca1+ cells in E14.5 FL and neither resolved into ESAMHi and ESAM−/Lo fractions. Expression profiles of Endoglin and Tie2 partially correlate with ESAM. The primitive ESAMHi fraction uniformly expressed high levels of Endoglin and Tie2, but many of the more differentiated ESAM−/Lo cells still retained the two markers. ESAM expression correlated well with HSC activity. Cells in the ESAMHi Rag1-ckitHi Sca1+ fraction formed more and larger colonies than those in the ESAM-/Lo Rag1-ckitHi Sca1+ fraction. Particularly, most CFU-Mix, primitive progenitors with both myeloid and erythroid potential, were found in the ESAMHi fraction. In limiting dilution stromal cell co-cultures, we found that 1 in 2.1 ESAMHi Rag1-ckitHi Sca1+ cells and 1 in 3.5 ESAM−/Lo Rag1-ckitHi Sca1+ cells gave rise to blood cells. However, while only 1 in 125 ESAM−/Lo Rag1-ckitHi Sca1+ cells were lymphopoietic under these conditions, 1 in 8 ESAMHi Rag1-ckitHi Sca1+ cells produced CD19+ B lineage cells. In long-term reconstituting assays, ESAMHi Rag1-ckitHi Sca1+ cells contributed highly to the multi-lineage recovery of lympho-hematopoiesis in recipients, but no chimerism was detected in mice transplanted with ESAM−/Lo Rag1-ckitHi Sca1+ cells. These results suggested that HSC in E14.5 FL are exclusively present in the ESAMHi fraction. Tie2+ c-kit+ lympho-hematopoietic cells of E10.5 AGM also expressed high levels of ESAM. Furthermore, ESAM expression in adult bone marrow was detected on primitive progenitors and cells in the side population within the Lin-ckitHi Sca1+ fraction. Interestingly, the expression was up-regulated in aged mice. Based on these observations, we conclude that ESAM marks HSC throughout life in mice. We also observed that many of human cord blood CD34+ CD38− cells express ESAM, suggesting potential application for the purification of human HSC.

Foxo3a Is Essential for the Quiescence and Self-Renewal of Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 442-442
Author(s):  
Kana Miyamoto ◽  
Atsushi Hirao ◽  
Kiyomi Y. Araki ◽  
Fumio Arai ◽  
Kazuhito Naka ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Quiescent State ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Functions

Abstract Hematopoietic stem cells (HSCs) are maintained in an undifferentiated quiescent state in bone marrow (BM). Quiescent stem cells show resistance to various stresses, suggesting that mechanisms for protection of HSC life from stress contribute to maintenance of self-renewal capacity through a whole life in animals. We hypothesized that a signaling pathway for regulating aging might be involved in stem cell functions. FOXO transcription factors belong to the forkhead family of transcriptional regulators characterized by a conserved DNA-binding domain termed “forkhead box”. In C.elegans, genetic analyses have revealed the existence of a conserved insulin-like signaling involved in longevity. Conservation of this pathways lead to speculation that forkhead transcriptional factor are involved in life span in mammals. It was known that active-state Foxo3a is localized in nucleus, and we found HSC-specific nuclear localization of Foxo3a by immunocytochemistric study, therefore we generated gene-targeted Foxo3a−/− mice to analyze roles of Foxo in HSC regulation. Peripheral blood count showed decreased number of red blood cells in Foxo3a−/− mice, but numbers of white blood cells and platelets were normal. In colony-forming assay, we detected the numbers and sizes of myeloid, erythroid and mixed colonies derived from Foxo3a−/− BM mononuclear cells were all normal. These results suggest that the proliferation and differentiation of Foxo3a−/− progenitors were normal. However, the number of colony-forming cells present in long-term culture of Foxo3a−/− c-kit+Sca-1+Lin− (KSL) cells with stroma was significantly reduced. The ability of Foxo3a−/− HSCs to support long-term reconstitution of hematopoiesis in a competitive transplantation assay was also impaired, indicating that self-renewal capacity of HSCs was defective in Foxo3a−/− mice. To understand the mechanisms of this phenotypes, we evaluated the cell cycle status using BrdU (5-bromodeoxyuridine) incorporation but found no difference in Foxo3a+/+ and Foxo3a−/− progenitor cells. To directly evaluate HSC quiescence in Foxo3a−/− mice, we stained CD34−KSL cells with Pyronin Y. Although most Foxo3a+/+ CD34−KSL cells stained negatively for Pyronin Y, a sizable Pyronin Y+ population was detected among Foxo3a−/− CD34−KSL cells, demonstrating that loss of Foxo3a leads to a defect in the maintenance of HSCs quiescence. Since p38MAPK is selectively activated by environmental stress, we evaluated the activation status of p38MAPK in Foxo3a+/+ and Foxo3a−/− HSCs. Frequency of phosphorylated p38MAPK+ cells in Foxo3a−/−CD34−KSL cells was significantly increased than that of Foxo3a+/+CD34−KSL cells. Our results suggest that Foxo3a−/− HSCs subjected to tangible stress in vivo. Finally, we investigated the sensitivity of Foxo3a−/− mice to weekly 5-fluorouracil treatment in vivo. Although 60% of Foxo3a+/+mice survived for at least 4 weeks post-injection, all Foxo3a−/− mice were dead in 4 weeks. It suggests that Foxo3a protects hematopoietic cells from destruction by cell cycle-dependent myelotoxic agent. Taken together, our results demonstrate that Foxo3a plays a pivotal role in maintaining HSC quiescence and stress resistance.

SCL regulates the Quiescence and the Long-Term Competence of Hematopoietic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2520-2520
Author(s):  
Julie Lacombe ◽  
Sabine Herblot ◽  
Shanti Rojas-Sutterlin ◽  
André Haman ◽  
Stephane Barakat ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Gene Dosage ◽  
Critical Role ◽  
Bhlh Transcription Factor ◽  
Hematopoietic Stem

Abstract Abstract 2520 Poster Board II-497 The life-long production of blood cells depends on the regenerative capacity of a rare bone marrow population, the hematopoietic stem cells (HSCs). In the adult, the majority of HSCs are quiescent while a large proportion of progenitors are more cycling. The state of quiescence in HSCs is reversible and these cells can be triggered into cycle by chemotoxic injuries, exposure to cytokines in vitro, as well as transplantation in vivo. SCL/TAL1 is a bHLH transcription factor that has a critical role in generating HSCs during development. However, the role of SCL in adult HSCs is still a matter of debate. In the present study, we took several approaches to address this question. Scl expression was monitored by quantitative PCR analysis in a population that contains adult long-term reconstituting HSCs (LT-HSCs) at a frequency of 20–50%: Kit+Sca+Lin-CD150+CD48-. RT-PCR results were confirmed by β-galactosidase staining of these cells in Scl-LacZ mice. We show that Scl is highly expressed in LT-HSC and that its expression correlates with quiescence, i.e. Scl levels decrease when LT-HSCs exit the G0 state. In order to assess stem cell function, we performed several transplantation assays with adult bone marrow cells in which SCL protein levels were decreased at least two-fold by gene targeting or by RNA interference. 1) The mean stem cell activity of HSCs transplanted at ∼1 CRU was two-fold decreased in Scl heterozygous (Scl+/−) mice. 2) In competitive transplantation, the contribution of Scl+/− cells to primitive populations as well mature cells in the bone marrow was significantly decreased 8 months after transplantation. 3) In secondary transplantation assays, Scl+/− HSCs were severely impaired in their ability to reconstitute secondary recipient in stem cells and progenitor populations and in almost all mature lineages. 4) Reconstitution of the stem cell pool by adult HSCs expressing Scl-directed shRNAs was significantly decreased compared to controls. We therefore conclude that SCL levels regulate HSC long term competence. Since Scl levels decrease when LT-HSCs exit the G0 state, we addressed the question whether the cell cycle state of LT-HSCs is sensitive to Scl gene dosage. We stained bone marrow cell populations with Hoechst and Pyronin Y. At steady state, percentage LT-HSCs in G1 fraction appears to be significantly increased in mice lacking one allele of Scl. Furthermore, a three-fold increase in G1 fraction was also observed when cells were infected with Scl-directed shRNA, suggesting that a decrease in Scl levels facilitates G0-G1 transition. At the molecular level, we show by chromatin immunoprecipitation that SCL occupies the Cdkn1a and Id1 loci. Furthermore, in purified Kit+Sca+Lin-CD150+CD48- cells, the expression levels of these two regulators of HSC cell cycle and long-term functions are sensitive to Scl gene dosage. Together, our observations suggest that SCL impedes G0-G1 transition in HSCs and regulates their long-term competence. Disclosures: No relevant conflicts of interest to declare.

scholarly journals NRF2 Activation Impairs Quiescence and Bone Marrow Reconstitution Capacity of Hematopoietic Stem Cells

2017 ◽  
Vol 37 (19) ◽  
Author(s):  
Shohei Murakami ◽  
Takuma Suzuki ◽  
Hideo Harigae ◽  
Paul-Henri Romeo ◽  
Masayuki Yamamoto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem ◽  
Cell Cycle Entry ◽  
Transient Activation ◽  
Nrf2 Activation

ABSTRACT Tissue stem cells are maintained in quiescence under physiological conditions but proliferate and differentiate to replenish mature cells under stressed conditions. The KEAP1-NRF2 system plays an essential role in stress response and cytoprotection against redox disturbance. To clarify the role of the KEAP1-NRF2 system in tissue stem cells, we focused on hematopoiesis in this study and used Keap1-deficient mice to examine the effects of persistent activation of NRF2 on long-term hematopoietic stem cells (LT-HSCs). We found that persistent activation of NRF2 due to Keap1 deficiency did not change the number of LT-HSCs but reduced their quiescence in steady-state hematopoiesis. During hematopoietic regeneration after bone marrow (BM) transplantation, persistent activation of NRF2 reduced the BM reconstitution capacity of LT-HSCs, suggesting that NRF2 reduces the quiescence of LT-HSCs and promotes their differentiation, leading to eventual exhaustion. Transient activation of NRF2 by an electrophilic reagent also promotes the entry of LT-HSCs into the cell cycle. Taken together, our findings show that NRF2 drives the cell cycle entry and differentiation of LT-HSCs at the expense of their quiescence and maintenance, an effect that appears to be beneficial for prompt recovery from blood loss. We propose that the appropriate control of NRF2 activity by KEAP1 is essential for maintaining HSCs and guarantees their stress-induced regenerative response.

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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2617-2617
Author(s):  
Fumio Arai ◽  
Kentaro Hosokawa ◽  
Yumiko Nojima ◽  
Toshio Suda
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Telomerase Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 2617 Hematopoietic 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.

scholarly journals Leptin receptor, a surface marker for a subset of highly engrafting long-term functional hematopoietic stem cells

Leukemia ◽  
2020 ◽  
Author(s):  
Thao Trinh ◽  
James Ropa ◽  
Arafat Aljoufi ◽  
Scott Cooper ◽  
Anthony Sinn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Leptin Receptor ◽  
Surface Marker ◽  
Hematopoietic Stem

scholarly journals Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 31-31
Author(s):  
Maria Rosa Lidonnici ◽  
Giulia Chianella ◽  
Francesca Tiboni ◽  
Matteo Barcella ◽  
Ivan Merelli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Gene Set Enrichment Analysis ◽  
Lineage Commitment ◽  
Erythroid Cell ◽  
Beta Thalassemia ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals Robo4 Plays a Role in Bone Marrow Homing and Mobilization, but Is Not Essential in the Long-Term Repopulating Capacity of Hematopoietic Stem Cells

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e50849 ◽  
Author(s):  
Yuko Goto-Koshino ◽  
Yumi Fukuchi ◽  
Fumi Shibata ◽  
Daichi Abe ◽  
Kana Kuroda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem
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