a-culture-method-to-maintain-quiescent-human-hematopoietic-stem-cells

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

National Center for Global Health and Medicine

Upon systemic bacterial infection, hematopoietic stem and progenitor cells (HSPCs) migrate to the periphery in order to supply a sufficient number of immune cells. Although pathogen-associated molecular patterns reportedly mediate HSPC activation, how HSPCs detect pathogen invasion in vivo remains elusive. Bacteria use the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) for a variety of activities. Here, we report that c-di-GMP comprehensively regulated both HSPCs and their niche cells through an innate immune sensor, STING, thereby inducing entry into the cell cycle and mobilization of HSPCs while decreasing the number and repopulation capacity of long-term hematopoietic stem cells. Furthermore, we show that type I interferon acted as a downstream target of c-di-GMP to inhibit HSPC expansion in the spleen, while transforming growth factor-β was required for c-di-GMP-dependent splenic HSPC expansion. Our results define machinery underlying the dynamic regulation of HSPCs and their niches during bacterial infection through c-di-GMP/STING signaling. 

Bacterial c-di-GMP affects hematopoietic stem/progenitors and their niches through STING.

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for their maintenance, proliferation, and differentiation. The BM niche is composed of nonhematopoietic and mature hematopoietic cells, including megakaryocytes (Mks). Thrombopoietin (Thpo) is a crucial cytokine produced by BM niche cells. However, the cellular source of Thpo, upon which HSCs primarily depend, is unclear. Moreover, no specific molecular pathway for the regulation of Thpo production in the BM has been identified. Here, we demonstrate that the membrane protein C-type lectin-like receptor-2 (CLEC-2) mediates the production of Thpo and other factors in Mks. Mice conditionally deleted for CLEC-2 in Mks (Clec2(MkΔ/Δ)) produced lower levels of Thpo in Mks. CLEC-2-deficient Mks showed down-regulation of CLEC-2-related signaling molecules Syk, Lcp2, and Plcg2. Knockdown of these molecules in cultured Mks decreased expression of Thpo. Clec2(MkΔ/Δ) mice exhibited reduced BM HSC quiescence and repopulation potential, along with extramedullary hematopoiesis. The low level of Thpo production may account for the decline in HSC potential in Clec2(MkΔ/Δ) mice, as administration of recombinant Thpo to Clec2(MkΔ/Δ) mice restored stem cell potential. Our study identifies CLEC-2 signaling as a novel molecular mechanism mediating the production of Thpo and other factors for the maintenance of HSCs.

CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow.

Hematopoietic stem/progenitor cells (HSPCs) play important roles in fighting systemic infection as they supply immune cells in a demand-adapted manner. Various mechanisms govern HSPC responses to infection, including cytokine signaling, niche function, and direct sensing of pathogen-derived molecules by HSPCs themselves. Here we review recent advances in our understanding of HSPC responses to infection and also consider newly identified STING-mediated machinery recognizing bacteria-derived cyclic dinucleotides. 

How hematopoietic stem/progenitors and their niche sense and respond to infectious stress.

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery.

p38α Activates Purine Metabolism to Initiate Hematopoietic Stem/Progenitor Cell Cycling in Response to Stress.

Polycomb repressive complex 2 (PRC2) participates in transcriptional repression through methylation of histone H3K27. The WD-repeat protein embryonic ectoderm development (EED) is a non-catalytic but an essential component of PRC2 and its mutations were identified in hematopoietic malignancies. To clarify the role(s) of EED in adult hematopoiesis and leukemogenesis, we generated Eed conditional knockout (Eed(Δ/Δ)) mice. Eed(Δ/Δ) mice died in a short period with rapid decrease of hematopoietic cells. Hematopoietic stem/progenitor cells (HSPCs) were markedly decreased with impaired bone marrow (BM) repopulation ability. Cell cycle analysis of HSPCs demonstrated increased S-phase fraction coupled with suppressed G0/G1 entry. Genes encoding cell adhesion molecules are significantly enriched in Eed(Δ/Δ) HSPCs, and consistently, Eed(Δ/Δ) HSPCs exhibited increased attachment to a major extracellular matrix component, fibronectin. Thus, EED deficiency increases proliferation on one side but promotes quiescence possibly by enhanced adhesion to the hematopoietic niche on the other, and these conflicting events would lead to abnormal differentiation and functional defect of Eed(Δ/Δ) HSPCs. In addition, Eed haploinsufficiency induced hematopoietic dysplasia, and Eed heterozygous mice were susceptible to malignant transformation and developed leukemia in cooperation with Evi1 overexpression. Our results demonstrated differentiation stage-specific and dose-dependent roles of EED in normal hematopoiesis and leukemogenesis.

Maintenance of the functional integrity of mouse hematopoiesis by EED and promotion of leukemogenesis by EED haploinsufficiency.

During steady-state conditions, hematopoietic stem cells (HSCs) maintain a quiescent status in the cell cycle. Upon infection or inflammation, bone marrow HSCs begin proliferating and generating differentiated hematopoietic cells via multi-lineage differentiation and self-renewal; this effect is partially due to the alteration of their surrounding microenvironment or niche. In addition, recent studies have revealed that the bone marrow niche critically contributes to abnormal hematopoiesis, including leukemogenesis. In this review, we discuss the recent advances in our understanding of HSC/niche functions and the regulatory machineries employed during homeostasis, stress hematopoiesis, or disease conditions.

Abnormal hematopoiesis and hematopoietic stem cell niche.

Hematopoietic stem cells (HSCs) are quiescent cells in the bone marrow niche and are relatively dependent on glycolytic ATP production. On the other hand, differentiated cells, including hematopoietic progenitor cells (HPCs), preferentially generate ATP via oxidative phosphorylation. However, it is unclear how cellular differentiation and the cell cycle status affect nutritional requirements and ATP production in HSCs and HPCs. Using a newly developed culture system, we demonstrated that survival of HPCs was strongly dependent on glucose, whereas quiescent HSCs survived for a certain duration without glucose. Among HPCs, granulocyte/monocyte progenitors (GMPs) were particularly dependent on glucose during proliferation. By monitoring the ATP concentration in live cells, we demonstrated that the ATP level was maintained for a short duration without glucose in HSCs, possibly due to their metabolic flexibility. In addition, HSCs exhibited low ATP turnover, whereas HPCs including GMPs demonstrated high ATP turnover and required efficient ATP production from glucose. These findings show that ATP turnover and nutritional requirements differ between HSCs and HPCs according to the cell cycle and differentiation status.

ATP turnover and glucose dependency in hematopoietic stem/progenitor cells are increased by proliferation and differentiation.

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions.

Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo.

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring  fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were -negative. Moreover, in some patients  was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph B cells but not bone marrow stem/progenitors.

Mammalian hematopoietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow. HSCs remain quiescent , unlike more differentiated progenitors, and enter the cell cycle rapidly after bone marrow injury or  culture. We have recently demonstrated the ability to maintain HSC quiescence  by mimicking the bone marrow microenvironment. Here, we provide a detailed protocol for keeping functional HSCs in the quiescent state . For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2019).

Protocol for the Maintenance of Quiescent Murine Hematopoietic Stem Cells.

Epigenetic regulation is essential for the maintenance of the hematopoietic system, and its deregulation is implicated in hematopoietic disorders. In this study, UTX, a demethylase for lysine 27 on histone H3 (H3K27) and a component of COMPASS-like and SWI/SNF complexes, played an essential role in the hematopoietic system by globally regulating aging-associated genes. Utx-deficient (UtxΔ/Δ) mice exhibited myeloid skewing with dysplasia, extramedullary hematopoiesis, impaired hematopoietic reconstituting ability, and increased susceptibility to leukemia, which are the hallmarks of hematopoietic aging. RNA-sequencing (RNA-seq) analysis revealed that Utx deficiency converted the gene expression profiles of young hematopoietic stem-progenitor cells (HSPCs) to those of aged HSPCs. Utx expression in hematopoietic stem cells declined with age, and UtxΔ/Δ HSPCs exhibited increased expression of an aging-associated marker, accumulation of reactive oxygen species, and impaired repair of DNA double-strand breaks. Pathway and chromatin immunoprecipitation analyses coupled with RNA-seq data indicated that UTX contributed to hematopoietic homeostasis mainly by maintaining the expression of genes downregulated with aging via demethylase-dependent and -independent epigenetic programming. Of note, comparison of pathway changes in UtxΔ/Δ HSPCs, aged muscle stem cells, aged fibroblasts, and aged induced neurons showed substantial overlap, strongly suggesting common aging mechanisms among different tissue stem cells.

UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy.

Hematopoietic stem cells (HSCs) and their progeny sustain lifetime hematopoiesis. Aging alters HSC function, number, and composition and increases risk of hematological malignancies, but how these changes occur in HSCs remains unclear. Signaling via p38MAPK has been proposed as a candidate mechanism underlying induction of HSC aging. Here, using genetic models of both chronological and premature aging, we describe a multimodal role for p38α, the major p38MAPK isozyme in hematopoiesis, in HSC aging. We report that p38α regulates differentiation bias and sustains transplantation capacity of HSCs in the early phase of chronological aging. However, p38α decreased HSC transplantation capacity in the late progression phase of chronological aging. Furthermore, co-deletion of p38α in mice deficient in Ataxia-telangiectasia mutated (Atm), a model of premature aging, exacerbated aging-related HSC phenotypes seen in Atm single mutant mice. Overall, these studies provide new insight into multiple functions of p38MAPK, which both promotes and suppresses HSC aging context-dependently.

p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts.

In diabetic pathology, insufficiency in β-cell mass unable to meet peripheral insulin demand and functional defects of individual β cells to produce insulin are often concurrently observed, collectively causing hyperglycemia. Here we show that the phosphorylation of ERK1/2 is significantly decreased in the islets of db/db mice as well as in those of a cohort of subjects with type 2 diabetes. In mice with abrogation of ERK signaling in pancreatic β cells through deletion of Mek1 and Mek2, glucose intolerance aggravates under high-fat diet-fed conditions due to insufficient insulin production with lower β-cell proliferation and reduced β-cell mass, while in individual β cells dampening of the number of insulin exocytosis events is observed, with the molecules involved in insulin exocytosis being less phosphorylated. These data reveal bifunctional roles for MEK/ERK signaling in β cells for glucose homeostasis, i.e., in regulating β-cell mass as well as in controlling insulin exocytosis in individual β cells, thus providing not only a novel perspective for the understanding of diabetes pathophysiology but also a potential clue for new drug development for diabetes treatment.

Article	Year
Bacterial c-di-GMP affects hematopoietic stem/progenitors and their niches through STING. Cell reports\| PubMed ID: 25843711	2015
CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow. The Journal of experimental medicine\| PubMed ID: 26552707	2015
How hematopoietic stem/progenitors and their niche sense and respond to infectious stress. Experimental hematology\| PubMed ID: 26646990	2016
p38α Activates Purine Metabolism to Initiate Hematopoietic Stem/Progenitor Cell Cycling in Response to Stress. Cell stem cell\| PubMed ID: 27345838	2016
Maintenance of the functional integrity of mouse hematopoiesis by EED and promotion of leukemogenesis by EED haploinsufficiency. Scientific reports\| PubMed ID: 27432459	2016
Abnormal hematopoiesis and hematopoietic stem cell niche. [Rinsho ketsueki] The Japanese journal of clinical hematology\| PubMed ID: 28978823	2017
ATP turnover and glucose dependency in hematopoietic stem/progenitor cells are increased by proliferation and differentiation. Biochemical and biophysical research communications\| PubMed ID: 31030941	2019
Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo. Cell reports\| PubMed ID: 31269436	2019
Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph B cells but not bone marrow stem/progenitors. Leukemia & lymphoma\| PubMed ID: 33106061	2021
Protocol for the Maintenance of Quiescent Murine Hematopoietic Stem Cells. STAR protocols\| PubMed ID: 33111112	2020
UTX maintains the functional integrity of the murine hematopoietic system by globally regulating aging-associated genes. Blood\| PubMed ID: 33174606	2021
OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy. Cell reports\| PubMed ID: 33406421	2021
p38α plays differential roles in hematopoietic stem cell activity dependent on aging contexts. The Journal of biological chemistry\| PubMed ID: 33745970	2021
MEK/ERK Signaling in β Cells Bifunctionally Regulates β-cell Mass and Glucose-stimulated Insulin-secretion Response to Maintain Glucose Homeostasis. Diabetes\| PubMed ID: 33906910	2021

Hiroshi Kobayashi

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