Early megakaryocyte lineage-committed progenitors in adult mouse bone marrow.

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors (MkPs) are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) mainly in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in adult mice. Single-cell colony assay and single-cell transplantation showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average 8 megakaryocytes per colony) than did previously reported MkPs. Single-cell RNA sequencing supported that HSCs give rise to MkPs through MgPs along a Mk differentiation pathway. Single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis showed that MgPs expressed Mk-related genes, but were transcriptionally heterogenous. Clonal culture of HSCs suggested that MgPs are not direct progeny of HSCs. We propose a differentiation model in which HSCs give rise to MgPs which then give rise to MkPs, supporting a classic model in which Mk-lineage commitment takes place at a late stage of differentiation.

Myelodysplastic syndromes are multiclonal diseases derived from hematopoietic stem and progenitor cells.

Myelodysplastic syndromes (MDS) are generally considered as a group of clonal diseases derived from hematopoietic stem cells, but a number of studies have suggested that they are derived from myeloid progenitor cells. We aimed to identify the cell of origin in MDS by single-cell analyses. Targeted single-cell RNA sequencing, covering six frequently mutated genes (U2AF1, SF3B1, TET2, ASXL1, TP53, and DNMT3A) in MDS, was developed and performed on individual cells isolated from the CD34+ and six lineage populations in the bone marrow of healthy donors (HDs) and patients with MDS. The detected mutations were used as clonal markers to define clones. By dissecting the distribution of clones in six lineages, the clonal origin was determined. We identified three mutations both in HDs and patients with MDS, termed clonal hematopoiesis (CH) mutations. We also identified fifteen mutations only detected in patients with MDS, termed MDS mutations. Clonal analysis showed that CH clones marked by CH mutations and MDS clones marked by MDS mutations were derived from hematopoietic stem cells as well as various hematopoietic progenitor cells. Most patients with MDS showed the chimeric state with CH clones and MDS clones. Clone size analysis suggested that CH mutations may not contribute to clonal expansion of MDS. In conclusion, MDS comprise multiple clones derived from hematopoietic stem and progenitor cells.

Multiple cells of origin in common with various types of mouse N-Myc acute leukemia.

Little is known regarding whether the cell of origin differs among different leukemia types. To address this fundamental issue, we determined the cell of origin in five distinct types of acute leukemia induced by N-Myc overexpression in mice. CD150+CD48-CD41-CD34-c-Kit+Sca-1+Lin- (KSL) (HSC1) cells, CD150-CD48-CD41-CD34-KSL (HSC2) cells, CD150+CD41+CD34-KSL (HPC1) cells, CD150+CD41+CD34+KSL (HPC2) cells, and CD150-CD41-CD34+KSL (HPC3) cells were purified from the bone marrow of adult C57BL/6 mice, transduced with the N-Myc retrovirus vector, and transplanted into lethally irradiated mice. B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and mixed phenotype acute leukemia (MPAL) developed from five populations. RNA sequencing data supported the phenotypical diagnoses of leukemia, except that AUL appeared transcriptionally close to T-ALL. Whole-genome sequencing revealed that retroviral integration sites were irrelevant to the leukemia types and that T-ALL and AML of MPAL shared the same integration site and many gene mutations, suggesting their common origin. Additionally, leukemic stem cells were identified in the KSL cell population, suggesting that the phenotypes of leukemic stem cells are irrelevant to leukemia types. This study provides experimental evidence for the similar and multiple cells of origin in acute leukemia.

Expansion of Quiescent Hematopoietic Stem Cells under Stress and Nonstress Conditions in Mice.

Hematopoietic stem cells (HSCs) are maintained in the quiescent state for protection from stress. How quiescent HSCs expand in vivo under stress and nonstress conditions, however, is poorly understood. Using the fluorescent ubiquitination-based cell cycle indicator (Fucci) mice, we analyzed quiescent and cycling HSCs in the bone marrow after transplantation and during development and aging. The cell cycle of HSCs in Fucci mice were analyzed by flow cytometry. Single-cell colony assays suggested that cycling cells were likely in the process of differentiation. Long-term competitive repopulation and limiting dilution assays revealed that given a higher frequency of functional HSCs in quiescent cells, durable self-renewal potential was greater in quiescent cells than cycling cells. In the bone marrow, functional HSC pool, represented by quiescent HSCs, was rapidly re-established by three weeks after transplantation, significantly expanded by three weeks of age in development, and gradually accumulated with aging. Single-cell RNA-sequencing with flow cytometric index sorting suggested that high levels of CD201 and Sca-1 expression and a low level of mitochondrial activity were associated with quiescent HSCs. A set of candidate quiescent genes in HSCs were also provided. This study implied that controlling quiescence in HSCs is important for their in vivo expansion and maintenance.

[Application and Research Progress of Lineage Tracing in Differentiation Mechanism of Hematopoietic Stem Cells--Review].

Hematopoietic stem cells (HSCs) reside at the top of the hierarchy and have the ability to differentiate to variety of hematopoietic progenitor cells (HPCs) or mature hematopoietic cells in each system. At present, the procress of HSC and HPC differentiating to the complete hematopoietic system under physiological and stressed conditions is poorly understood. In vivo lineage tracing is a powerful technique that can mark the individual cells and identify the differentiation pathways of their daughter cells, it takes as a strong technical system to research HSC. Traditional lineage tracing studies mainly rely on imaging techniques with fluorescent dyes and nucleic acid analogs. Recently, newly cell tracing technologies have been invented, and the combination of clonal tracing and DNAsequencing technologies have provided a new perspective on cell state, cell fate, and lineage commitment at the single cell level. In this review, these new tracing methods were introduce and discuss, and their advantages over traditional methods in the study of hematopoiesis were summarized briefly.

Gene knockout in highly purified mouse hematopoietic stem cells by CRISPR/Cas9 technology.

The CRISPR/Cas9 system has been used for genome editing of human and mouse cells. In this study, we established a protocol for gene knockout (KO) in mouse hematopoietic stem cells (HSCs). HSCs were highly purified from the bone marrow of tamoxifen-treated Cas9-EGFP/Cre-ER transgenic mice, maintained in serum-free polyvinyl alcohol culture with cytokines, lentivirally transduced with sgRNA-Crimson, and transplanted into lethally irradiated mice with competitor cells. Previous studies of Pax5 KO mice have shown B cell differentiation block. To verify our KO HSC strategy, we deleted Pax5 gene in 600 CD201+CD150+CD48-c-Kit+Sca-1+Lin- cells (HSC1 cells), highly enriched in myeloid-biased HSCs, and CD201+CD150-CD48- c-Kit+Sca-1+Lin- cells (HSC2 cells), highly enriched in lymphoid-biased HSCs. As predicted, both Pax5 KO HSC1 and HSC2 cells showed few B cells in the peripheral blood and the accumulation of pro-B cells in the bone marrow of recipient mice. Our data suggesetd that myeloid-biased and lymphoid-biased HSCs share a common B cell differentiation pathway. This population-specific KO strategy will find its applications for gene editing in a varity of somatic cells, particuarly rare stem and progenitor cells from different tissues.

Analysis and Isolation of Mouse Leukemic Stem Cells.

Flow cytometry has been widely used in basic and clinical research for analysis of a variety of normal and malignant cells. Hematopoietic stem cells (HSCs) and leukemic stem cells (LSCs) can be highly purified by flow cytometry. Isolated HSCs and LSCs can be functionally identified by transplantation assays and can also be studied at the molecular level. Here we describe the flow cytometry methods for analysis and isolation of mouse HSCs and LSCs.

Granulocyte colony-stimulating factor directly acts on mouse lymphoid-biased but not myeloid-biased hematopoietic stem cells.

Granulocyte colony-stimulating factor (G-CSF) is widely used in clinical settings to mobilize hematopoietic stem cells (HSCs) into the circulation for HSC harvesting and transplantation. However, whether G-CSF directly stimulates HSCs to change their cell cycle state and fate is controversial. HSCs are a heterogeneous population consisting of different types of HSCs, such as myeloid-biased HSCs and lymphoid-biased HSCs. We hypothesized that G-CSF has different effects on different types of HSCs. To verify this, we performed serum-free single-cell culture and competitive repopulation with cultured cells. Single highly purified HSCs and hematopoietic progenitor cells (HPCs) were cultured with stem cell factor (SCF), SCF + G-CSF, SCF + granulocyte/macrophage (GM)-CSF, or SCF + thrombopoietin (TPO) for 7 days. Compared with SCF alone, SCF + G-CSF increased the number of divisions of cells from the lymphoid-biased HSC-enriched population but not that of cells from the My-bi HSC-enriched population. SCF + G-CSF enhanced the level of reconstitution of lymphoid-biased HSCs but not that of myeloid-biased HSCs. Clonal transplantation assay also showed that SCF + G-CSF did not increase the frequency of myeloid-biased HSCs. These data showed that G-CSF directly acted on lymphoid-biased HSCs but not myeloid-biased HSCs. Our study also revised the cytokine network at early stages of hematopoiesis: SCF directly acted on myeloid-biased HSCs; TPO directly acted on myeloid-biased HSCs and lymphoid-biased HSCs; and GM-CSF acted only on HPCs. Early hematopoiesis is controlled differentially and sequentially by a number of cytokines.

Lymphoid-biased hematopoietic stem cells and myeloid-biased hematopoietic progenitor cells have radioprotection activity.

Radioprotection was previously considered as a function of hematopoietic stem cells (HSCs). However, recent studies have reported its activity in hematopoietic progenitor cells (HPCs). To address this issue, we compared the radioprotection activity in 2 subsets of HSCs (nHSC1 and 2 populations) and 4 subsets of HPCs (nHPC1-4 populations) of the mouse bone marrow, in relation to their in vitro and in vivo colony-forming activity. Significant radioprotection activity was detected in the nHSC2 population enriched in lymphoid-biased HSCs. Moderate radioprotection activity was detected in nHPC1 and 2 populations enriched in myeloid-biased HPCs. Low radioprotection activity was detected in the nHSC1 enriched in myeloid-biased HSCs. No radioprotection activity was detected in the nHPC3 and 4 populations that included MPP4 (LMPP). Single-cell colony assay combined with flow cytometry analysis showed that the nHSC1, nHSC2, nHPC1, and nHPC2 populations had the neutrophils/macrophages/erythroblasts/megakaryocytes (nmEMk) differentiation potential whereas the nHPC3 and 4 populations had only the nm differentiation potential. Varying day 12 spleen colony-forming units (day 12 CFU-S) were detected in the nHSC1, nHSC2, and nHPC1-3 populations, but very few in the nHPC4 population. These data suggested that nmEMk differentiation potential and day 12 CFU-S activity are partially associated with radioprotection activity. Reconstitution analysis showed that sufficient myeloid reconstitution around 12 to 14 days after transplantation was critical for radioprotection. This study implied that radioprotection is specific to neither HSC nor HPC populations, and that lymphoid-biased HSCs and myeloid-biased HPCs as populations play a major role in radioprotection.

[Hematopoietic Stem Cells Differentiate into the Megakaryocyte Lineage--Review].

Abstract　　Hematopoietic stem cells are able to self-renewal and differentiate to all blood lineages. With the development of new technologies, recent studies have proposed the revised versions of hematopoiesis. In the classical model of hematopoietic differentiation, HSCs were located at the apex of hematopoietic hierarchy. During differentiation process, HSCs progressively lose self-renewal potential to be commited to progenitors with restricted differentiation potential. For instance, HSCs first give rise to multipotent progenitor cells, then produce bipotent and unipotent progenitors, and finally differentiate to mature blood cells. For the differentiation of megakaryocytes, common myeloid progenitors derived from HSCs give rise to megakaryocyte-erythrocyte progenitors and then develop to megakaryocytes. However, recent results show that megakaryocytes can be directly generated from HSCs without multipotent or bipotent phases. Alternatively, platelet-biased HSCs produce megakaryocyte progenitors. In this article, recent advances in the hematopoiesis and megakaryocyte differentiation pathway are reviewed.

Differentiation of transplanted haematopoietic stem cells tracked by single-cell transcriptomic analysis.

How transplanted haematopoietic stem cells (HSCs) behave soon after they reside in a preconditioned host has not been studied due to technical limitations. Here, using single-cell RNA sequencing, we first obtained the transcriptome-based classifications of 28 haematopoietic cell types. We then applied them in conjunction with functional assays to track the dynamic changes of immunophenotypically purified HSCs in irradiated recipients within the first week after transplantation. Based on our transcriptional classifications, most homed HSCs in bone marrow and spleen became multipotent progenitors and, occasionally, some HSCs gave rise to megakaryocytic-erythroid or myeloid precursors. Parallel in vitro and in vivo functional experiments supported the paradigm of robust differentiation without substantial HSC expansion during the first week. Therefore, this study uncovers the previously inaccessible kinetics and fate choices of transplanted HSCs in myeloablated recipients at early stage, with implications for clinical applications of HSCs and other stem cells.

[Research Advance on In Vitro Generation of Human Hematopoietic Stem Cells for Transplantation--Review].

Abstract　　Currently, hematopoietic stem cell (HSC) transplantation is widely used in the therapy of hematological malignancies, non-malignant refractory anemia, genetic diseases and certain tumors with satisfactory therapeutic efficacy. HSC sources used for transplantation include bone marrow, mobilized peripheral blood and neonate umbilical cord blood. However, for many patients, sufficient number of human leukocyte antigen (HLA) -matched HSC cannot be found for transplantation, because the number of HSC in these tissues is small and HLA-identical donors are rare. Thus, in vitro generation of HSC has recently been focused. At present, the origin of HSC is hPSC, including hESC and hiPSC, which is worth to be the new origin of HSC transplantation. However, to generate functional hematopoietic stem cells which have efficient multi-lineage differentiation and in vivo engraftment potentials still is a big challenge to be confronted. In this review, the recent technical progress in HSC generation is summarizd, and the problems to be solved and new challenges to be confronted were discussed.

Lineage marker expression on mouse hematopoietic stem cells.

Whether hematopoietic stem cells (HSCs) express lineage markers is controversial. In this study, we highly purified HSCs from the adult bone marrow of C57BL/6 mice and examined their gene expression and reconstitution potential. We first focused on the integrin family. Single-cell reverse transcription polymerase chain reaction revealed that the expression of ItgaM/Itgb2 (Mac-1) and Itga2b/Itgb3 (CD41/CD61) gradually increased along HSC differentiation, whereas Itga4, Itga5, Itga6, and ItgaV (CD51) together with Itgb1 were highly expressed in both HSCs and hematopoietic progenitor cells (HPCs). We next fractionated HSCs based on their expression of Mac-1, CD41, and CD51 by flow cytometry. We detected Mac-negative and Mac-low, but not Mac-high cells, in the HSC population. We also detected CD41-negative, -low, and -high cells in the HSC population. Competitive repopulation revealed that Mac-1-negative and -low HSCs were functionally similar, and CD41-negative and -low HSCs were functionally similar, at the single-cell level, but CD41-high HSCs were not detectable. We then found that the selection of Mac-1-negative HSCs or CD41-negative HSCs had no advantage in HSC purification. We moreover found that HSCs expressed more CD51 than CD41, and HPCs expressed more CD41 than CD51, suggesting that CD51 expression was gradually replaced by CD41 expression during megakaryocyte differentiation. We concluded that low levels of Mac-1 and CD41 expression are irrelevant to the self-renewal and differentiation potentials in HSCs.

Mouse acute leukemia develops independent of self-renewal and differentiation potentials in hematopoietic stem and progenitor cells.

The cell of origin, defined as the normal cell in which the transformation event first occurs, is poorly identified in leukemia, despite its importance in understanding of leukemogenesis and improving leukemia therapy. Although hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) were used for leukemia models, whether their self-renewal and differentiation potentials influence the initiation and development of leukemia is largely unknown. In this study, the self-renewal and differentiation potentials in 2 distinct types of HSCs (HSC1 [CD150+CD41-CD34-Lineage-Sca-1+c-Kit+ cells] and HSC2 [CD150-CD41-CD34-Lineage-Sca-1+c-Kit+ cells]) and 3 distinct types of HPCs (HPC1 [CD150+CD41+CD34-Lineage-Sca-1+c-Kit+ cells], HPC2 [CD150+CD41+CD34+Lineage-Sca-1+c-Kit+ cells], and HPC3 [CD150-CD41-CD34+Lineage-Sca-1+c-Kit+ cells]) were isolated from adult mouse bone marrow, and examined by competitive repopulation assay. Then, cells from each population were retrovirally transduced to initiate MLL-AF9 acute myelogenous leukemia (AML) and the intracellular domain of NOTCH-1 T-cell acute lymphoblastic leukemia (T-ALL). AML and T-ALL similarly developed from all HSC and HPC populations, suggesting multiple cellular origins of leukemia. New leukemic stem cells (LSCs) were also identified in these AML and T-ALL models. Notably, switching between immunophenotypical immature and mature LSCs was observed, suggesting that heterogeneous LSCs play a role in the expansion and maintenance of leukemia. Based on this mouse model study, we propose that acute leukemia arises from multiple cells of origin independent of the self-renewal and differentiation potentials in hematopoietic stem and progenitor cells and is amplified by LSC switchover.

[Emerging role of Mitochondria in Maintenance of Hematopoietic Stem Cells--Review].

Mitochondria are double-membrane organelles existing only in eukaryotic cells. Mitochondria perform various important functions，such as producing energy，regulating signal transduction，and contributing to stress response. Recent studies have highlighted an important role of mitochondria in the determination of hematopoietic stem cells （HSC） fate. Limited biogenesis or timely clearance of mitochondria is an important way against oxidative stress，which favors the quiescence of HSC. Accumulation of mitochondria may lead to proliferation of HSC，even the aging of HSC. Mitochondrial signaling regulates Ca2+ concentration，which is essential for HSC differentiation. This review summarizes the current findings of the mitochondrial roles in HSC quiescence，self-renewal，lineage differentiation and aging.

Interleukin-12 supports in vitro self-renewal of long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) self-renew or differentiate through division. Cytokines are essential for inducing HSC division, but the optimal cytokine combination to control self-renewal of HSC in vitro remains unclear. In this study, we compared the effects of interleukin-12 (IL-12) and thrombopoietin (TPO) in combination with stem cell factor (SCF) on in vitro self-renewal of HSCs. Single-cell assays were used to overcome the heterogeneity issue of HSCs, and serum-free conditions were newly established to permit reproduction of data. In single-cell cultures, CD150+CD48-CD41-CD34-c-Kit+Sca-1+lineage- HSCs divided significantly more slowly in the presence of SCF+IL-12 compared with cells in the presence of SCF+TPO. Serial transplantation of cells from bulk and clonal cultures revealed that TPO was more effective than IL-12 at supporting in vitro self-renewal of short-term (<6 months) HSCs, resulting in a monophasic reconstitution wave formation, whereas IL-12 was more effective than TPO at supporting the in vitro self-renewal of long-term (>6 months) HSCs, resulting in a biphasic reconstitution wave formation. The control of division rate in HSCs appeared to be crucial for preventing the loss of self-renewal potential from their in vitro culture.

Successful ex vivo expansion of mouse hematopoietic stem cells.

Ex vivo expansion of hematopoietic stem cells (HSCs) is considered the holy grail in stem cell biology and therapy, as it has long been difficult to make this procedure possible. Yamazaki's research team has established new, polyvinyl alcohol-based culture conditions and shown a significant expansion of mouse HSCs from a small number of cells after a month of culture. Surprisingly, expanded HSCs were able to reconstitute unconditioned normal mice. There is generally a technical concern in limiting dilution assay to estimate a fold-expansion of HSCs. But, this work paves the way toward expansion of human HSCs useful for transplantation medicine.

TGF-ß1 Negatively Regulates the Number and Function of Hematopoietic Stem Cells.

Transforming growth factor ß1 (TGF-ß1) plays a role in the maintenance of quiescent hematopoietic stem cells (HSCs) in vivo. We asked whether TGF-ß1 controls the cell cycle status of HSCs in vitro to enhance the reconstitution activity. To examine the effect of TGF-ß1 on the HSC function, we used an in vitro culture system in which single HSCs divide with the retention of their short- and long-term reconstitution ability. Extensive single-cell analyses showed that, regardless of its concentration, TGF-ß1 slowed down the cell cycle progression of HSCs but consequently suppressed their self-renewal potential. Cycling HSCs were not able to go back to quiescence with TGF-ß1. This study revealed a negative role of TGF-ß1 in the regulation of the HSC number and reconstitution activity.

Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress.

Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.