Understanding the “SMART” features of hematopoietic stem cells and beyond

Science China Life Sciences - Since the huge success of bone marrow transplantation technology in clinical practice, hematopoietic stem cells (HSCs) have become the gold standard for defining the...     

A polarized anchor for hematopoietic stem cells: Synapse between stem cells and their niche?

Multipotent hematopoietic stem cells are maintained by the bone marrow niche, but how niche-derived membrane-bound stem cell factor (mSCF) regulates HSCs remains unclear. In this issue, Hao et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010118) describe that mSCF, synergistically with VCAM-1, induces large, polarized protrusions that serve as anchors for HSCs to their niche.

Hematopoietic stem cells (HSCs) generate all blood and immune cells throughout life via self-renewal and multilineage differentiation within the bone marrow niche. HSCs are the basis for bone marrow transplantation, saving thousands of lives yearly. The bone marrow niche often serves as a paradigm for studying stem cell biology. In addition, elucidating the underlying mechanism in the niche helps devise strategies to expand functional HSCs for clinical use. Within the niche, leptin receptor–positive perisinusoidal stromal cells and endothelial cells are the major source of essential cytokines for HSC maintenance, including vascular cell adhesion molecule 1 (VCAM-1) and stem cell factor (SCF; 1, 2). Locally produced soluble and membrane-bound cytokines preserve the unique localization and anchorage of HSCs to stromal cells within their niche. Consistent with this notion, mouse genetic data have shown that membrane-bound SCF (mSCF) is important for HSC maintenance in vivo (3). However, given that both soluble and membrane-bound forms of SCF can engage with the cognate cKIT receptors, the mechanisms by which mSCF sustains HSCs function in vivo remain elusive. Likewise, it is unclear why the expansion and maintenance of HSCs ex vivo by adding SCF to culture as an either soluble or immobilized form has only been achieved with limited success.In this issue, Hao et al. addressed this question by using a supported lipid bilayer (SLB) system to model the interaction between HSCs and membrane-bound cytokines, including SCF (4). SLBs present an advantage over conventional immobilization methods; they allow the lateral mobility of membrane-bound proteins and clustering of receptors and signaling complexes, thus resembling the lipid bilayer of plasma membrane in vivo. Focusing on HSC cytokines that may be presented as membrane-bound forms in the bone marrow niche, the authors performed an imaging screen in vitro using SLBs and found that mSCF but not soluble SCF (sSCF) induced mSCF/cKIT clustering and the formation of membrane protrusions on HSCs. While mSCF alone was sufficient to promote cell protrusions, HSCs required both mSCF and VCAM-1 for large, polarized protrusions. They followed HSCs at different time points after exposure to VCAM-1 and mSCF by scanning electron microscopy and observed that HSCs first formed diffuse mSCF clusters and multifocal thin protrusions and then proceeded to a polarized, clustered morphology with larger and thicker protrusions. Using a controlled sheer stress device, Hao et al. showed that these polarized protrusions had a functional consequence on the adhesion strength of HSCs. mSCF and VCAM-1 dramatically increased the adhesion of HSCs to SLB compared with VCAM-1 or mSCF alone. Interestingly, the effect was more prominent in HSCs compared with their immediate downstream progenies, multipotent progenitors. This phenotype was also specific to ligands presented on SLB because the effect was canceled when the cytokines were directly immobilized onto the glass surface. Then, they had a close look into the cytoskeletal organization of HSCs in the presence of both mSCF and VCAM-1 on SLB. They found that F-actin and myosin IIa concentrated at the protrusion, which led them to speculate that the cytoskeleton remodeling mediates the formation of the polarized morphology. Indeed, chemical inhibitors blocking myosin contraction, actin polymerization, or Rho-associated protein kinase disrupted the formation of the large and polarized protrusion. The authors noted that phosphatidylinositol 3-kinase (PI3K) also localized with mSCF/cKIT clusters, so they further assessed the contribution of the PI3K/Akt pathway to the polarized morphology of HSCs by using total internal reflection fluorescence microscopy and PI3K and Akt chemical inhibitors. PI3K/Akt activation contributed downstream of the mSCF–VCAM-1 synergy to regulating HSC cell adhesion and polarized mSCF/cKIT distribution. In addition, PI3K signaling enhanced the nuclear retention of FOXO3a, a crucial factor for HSC self-renewal; this enhancement was induced by mSCF but lessened by sSCF. Intriguingly, sSCF also competed with mSCF and abrogated the effect of the mSCF–VCAM-1 synergy on polarized protrusion formation. However, whether and how PI3K transmits the mSCF–VCAM-1 synergy into proliferation or quiescence cues in HSCs requires further investigation. Taken together, these data suggest that mSCF and VCAM-1 synergize to induce polarized protrusions on HSCs, which regulates their adhesion to the niche (Fig. 1). These protrusions share many features with the immunological synapse (5), which points toward the existence of a similar model for stem cells, “stem cell synapse,” where HSCs interact with and receive a variety of signals from their niche cells.Open in a separate windowFigure 1.VCAM-1 and mSCF synergistically promote the formation of polarized protrusions (stem cell synapse) on HSCs. (A and B) VCAM-1 or mSCF alone does not induce apparent polarized morphology on HSCs. The signaling and adhesion of HSCs to the niche is not at its full potential. (C) VCAM-1 and mSCF together induce robust receptor clustering on HSCs, optimal signaling, and strong adhesion. (D) sSCF can competitively disrupt the polarized protrusions on HSCs. The figure was created with BioRender.com.While the study by Hao et al. sheds light on how niche signals, particularly mSCF, regulate HSCs, several outstanding questions remain. First, even though many hematopoietic cells express cKIT (some of them even express higher levels than HSCs), HSCs respond to mSCF + VCAM-1 the strongest by recruiting the most mSCF to clusters. What is the specific mechanism in HSCs underlying this specificity? Second, SCF is produced both as mSCF and sSCF in vivo, through alternative splicing and proteolytic cleavage; if mSCF is mainly responsible for anchoring HSCs in the niche, what is the function of sSCF in vivo? Does sSCF modulate the available pool of mSCF? Third, robust maintenance of HSCs in culture has been challenging. HSCs can be maintained in a system composed of sSCF, thromopoietin (TPO), fibronectin, and polyvinyl alcohol (6). Tethering cytokines to SLB elicits more physiological response from HSCs compared with soluble cytokines or direct immobilization. Does SLB improve maintenance of HSCs in in vitro culture? Fourth, some cytokines, such as TPO, act on HSCs in a long-range manner (7). How do these systemic cytokines induce robust signaling in HSCs? Do they participate in the stem cell synapse even if they are not the initiators? Finally, do stem cells and their niche interact by forming similar synapses in other stem cell systems? Answering these questions will deepen our understanding of the stem cell niche and help integrate the niche component into potential, more successful applications in regenerative medicine.     

Transient activation of hematopoietic stem and progenitor cells by IFNγ during acute bacterial infection

How hematopoietic stem cells (HSCs) respond to inflammatory signals during infections is not well understood. Our studies have used a murine model of ehrlichiosis, an emerging tick-born disease, to address how infection impacts hematopoietic function. Infection of C57BL/6 mice with the intracellular bacterium, Ehrlichia muris, results in anemia and thrombocytopenia, similar to what is observed in human ehrlichiosis patients. In the mouse, infection promotes myelopoiesis, a process that is critically dependent on interferon gamma (IFNγ) signaling. In the present study, we demonstrate that E. muris infection also drives the transient proliferation and expansion of bone marrow Lin-negative Sca-1(+) cKit(+) (LSK) cells, a population of progenitor cells that contains HSCs. Expansion of the LSK population in the bone marrow was associated with a loss of dormant, long-term repopulating HSCs, reduced engraftment, and a bias towards myeloid lineage differentiation within that population. The reduced engraftment and myeloid bias of the infection-induced LSK cells was transient, and was most pronounced on day 8 post-infection. The infection-induced changes were accompanied by an expansion of more differentiated multipotent progenitor cells, and required IFNγ signaling. Thus, in response to inflammatory signals elicited during acute infection, HSCs can undergo a rapid, IFNγ-dependent, transient shift from dormancy to activity, ostensibly, to provide the host with additional or better-armed innate cells for host defense. Similar changes in hematopoietic function likely underlie many different infections of public health importance.     

Long-term self-renewal of naïve neural stem cells in a defined condition

During embryonic development, neural stem cells (NSCs) emerge as early as the neural plate stage and give rise to the nervous system. Early-stage NSCs express Sry-related-HMG box-1 (Sox1) and are biased towards neuronal differentiation. However, long-term maintenance of early-stage NSCs in vitro remains a challenge. Here, we report development of a defined culture condition for the long-term maintenance of Sox1-positive early-stage mouse NSCs. The proliferative ability of these Sox1-positive NSCs was confirmed by clonal propagation. Compared to the NSCs cultured using the traditional culture condition, the long-term self-renewing Sox1-positive NSCs efficiently differentiate into neurons and exhibit an identity representative of the anterior and midbrain regions. These early-stage Sox1-positive NSCs could also be switched to late-stage NSCs by being cultured with bFGF/EGF, which can then differentiate into astrocytes and oligodendrocytes. The long-term self-renewing Sox1-positive NSCs were defined as naïve NSCs, based on their high neuronal differentiation capacity and anterior regional identity. This culture condition provides a robust platform for further dissection of the NSC self-renewal mechanism and promotes potential applications of NSCs for cell-based therapy on nervous system disorders.     

Impact of IFN-β and LIF overexpression on human adipose-derived stem cells properties

Adipose-derived stem cells (ADSCs) are a subset of mesenchymal stem cells that their therapeutic effects in various diseases make them an interesting tool in cell therapy. In the current study, we aimed to overexpress interferon-β (IFN-β) and leukemia inhibitory factor (LIF) cytokines in human ADSCs to evaluate the impact of this overexpression on human ADSCs properties. Here, we designed a construct containing IFN-β and LIF and then, transduced human adipose-derived stem cells (hADSCs) by this construct via a lentiviral vector (PCDH-513B). We assessed the ability of long-term expression of the transgene in transduced cells by western blot analysis and enzyme-linked immunosorbent assay techniques on Days 15, 45, and 75 after transduction. For the evaluation of stem cell properties, flow cytometry and differentiation assays were performed. Finally, the MTT assay was done to assess the proliferation of transduced cells compares to controls. Our results showed high-efficiency transduction with highest expression rates on Day 75 after transduction which were 70 pg/ml for IFN-β and 77.9 pg/ml for LIF in comparison with 25.60 pg/ml and 27.63 pg/ml, respectively, in untransduced cells (p = .0001). Also, transduced cells expressed a high level of ADSCs surface markers and successfully differentiated into adipocytes, chondrocytes, neural cells, and osteocytes besides the preservation rate of proliferation near untreated cells (p = .88). All in all, we successfully constructed an hADSC population stably overexpressed IFN-β and LIF cytokines. Considering the IFN-β and LIF anti-inflammatory and neuroprotective effects as well as immune-regulatory properties of hADSCs, the obtained cells of this study could be subjected for further evaluations in experimental autoimmune encephalomyelitis mice model.     

TGF-β family signaling in stem cells

Background

The diversity of cell types and tissue types that originate throughout development derives from the differentiation potential of embryonic stem cells and somatic stem cells. While the former are pluripotent, and thus can give rise to a full differentiation spectrum, the latter have limited differentiation potential but drive tissue remodeling. Additionally cancer tissues also have a small population of self-renewing cells with stem cell properties. These cancer stem cells may arise through dedifferentiation from non-stem cells in cancer tissues, illustrating their plasticity, and may greatly contribute to the resistance of cancers to chemotherapies.

Scope of review

The capacity of the different types of stem cells for self-renewal, the establishment and maintenance of their differentiation potential, and the selection of differentiation programs are greatly defined by the interplay of signaling molecules provided by both the stem cells themselves, and their microenvironment, the niche. Here we discuss common and divergent roles of TGF-β family signaling in the regulation of embryonic, reprogrammed pluripotent, somatic, and cancer stem cells.

Major conclusions

Increasing evidence highlights the similarities between responses of normal and cancer stem cells to signaling molecules, provided or activated by their microenvironment. While TGF-β family signaling regulates stemness of normal and cancer stem cells, its effects are diverse and depend on the cell types and physiological state of the cells.

General significance

Further mechanistic studies will provide a better understanding of the roles of TGF-β family signaling in the regulation of stem cells. These basic studies may lead to the development of a new therapeutic or prognostic strategies for the treatment of cancers. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Different roles of TGF-β in the multi-lineage differentiation of stem cells

Stem cells are a population of cells that has infinite or long-term self-renewal ability and can produce various kinds of descendent cells.Transforming growth factor β(TGF-β) family is a superfamily of growth factors,including TGF-β1,TGF-β2 and TGF-β3,bone morphogenetic proteins,activin/inhibin,and some other cytokines such as nodal,which plays very important roles in regulating a wide variety of biological processes,such as cell growth,differentiation,cell death.TGF-β,a pleiotropic cytokine,has been proved to be differentially involved in the regulation of multi-lineage differentiation of stem cells,through the Smad pathway,non-Smad pathways including mitogen-activated protein kinase pathways,phosphatidylinositol-3-kinase/AKT pathways and Rholike GTPase signaling pathways,and their cross-talks.For instance,it is generally known that TGF-β promotes the differentiation of stem cells into smooth muscle cells,immature cardiomyocytes,chondrocytes,neurocytes,hepatic stellate cells,Th17 cells,and dendritic cells.However,TGF-β inhibits the differentiation of stem cells into myotubes,adipocytes,endothelial cells,and natural killer cells.Additionally,TGF-β can provide competence for early stages of osteoblastic differentiation,but at late stages TGF-β acts as an inhibitor.The three mammalian isoforms(TGF-β1,2 and 3) have distinct but overlapping effects on hematopoiesis.Understanding the mechanisms underlying the regulatory effect of TGF-β in the stem cell multi-lineage differentiation is of importance in stem cell biology,and will facilitate both basic research and clinical applications of stem cells.In this article,we discuss the current status and progress in our understanding of different mechanisms by which TGF-β controls multi-lineage differentiation of stem cells.     

Mathematical study on kinetics of hematopoietic stem cells – theoretical conditions for successful transplantation

Numerous haematological diseases occur due to dysfunctions during homeostasis processes of blood cell production. Haematopoietic stem cell transplantation (HSCT) is a therapeutic option for the treatment of haematological malignancy and congenital immunodeficiency. Today, HSCT is widely applied as an alternative method to bone marrow transplantation; however, HSCT can be a risky procedure because of potential side effects and complications after transplantations. Although an optimal regimen to achieve successful HSCT while maintaining quality of life is to be developed, even theoretical considerations such as the evaluations of successful engraftments and proposals of clinical management strategies have not been fully discussed yet.

In this paper, we construct and investigate mathematical models that describe the kinetics of hematopoietic stem cell self-renewal and granulopoiesis under the influence of growth factors. Moreover, we derive theoretical conditions for successful HSCT, primarily on the basis of the idea that the basic reproduction number R ₀ represents a threshold condition for a population to successfully grow in a given steady-state environment. Successful engraftment of transplanted haematopoietic stem cells (HSCs) is subsequently ensured by employing a concept of dynamical systems theory known as ‘persistence’. On the basis of the implications from the modelling study, we discuss how the conditions derived for a successful HSCT are used to link to experimental studies.     

Impact of T cells on hematopoietic stem and progenitor cell function: Good guys or bad guys?

When hematopoietic stem and progenitor cells(HSPC)are harvested for transplantation, either from the bone marrow or from mobilized blood, the graft contains a significant number of T cells. It is these T cells that are the major drivers of graft-vs-host disease(Gv HD). The risk for Gv HD can simply be reduced by the removal of these T cells from the graft. However, this is not always desirable, as this procedure also decreases the engraftment of the transplanted HSPCs and, if applicable, a graft-vs-tumor effect. This poses an important conundrum in the field: T cells act as a double-edged sword upon allogeneic HSPC transplantation, as they support engraftment of HSPCs and provide anti-tumor activity, but can also cause Gv HD. It has recently been suggested that T cells also enhance the engraftment of autologous HSPCs, thus supporting the notion that T cells and HSPCs have an important functional interaction that is highly beneficial, in particular during transplantation. The underlying reason on why and how T cells contribute to HSPC engraftment is still poorly understood. Therefore, we evaluate in this review the studies that have examined the role of T cells during HSPC transplantation and the possible mechanisms involved in their supporting function. Understanding the underlying cellular and molecular mechanisms can provide new insight into improving HSPC engraftment and thus lower the number of HSPCs required during transplantation. Moreover, it could provide new avenues to limit the development of severe Gv HD, thus making HSPC transplantations more efficient and ultimately safer.     

Neonatal agonism of ERβ impairs male reproductive behavior and attractiveness

The organization of the developing male rodent brain is profoundly influenced by endogenous steroids, most notably estrogen. This process may be disrupted by estrogenic endocrine disrupting compounds (EDCs) resulting in altered sex behavior and the capacity to attract a mate in adulthood. To better understand the relative role each estrogen receptor (ER) subtype (ERα and ERβ) plays in mediating these effects, we exposed male Long Evans rats to estradiol benzoate (EB, 10 μg), vehicle, or agonists specific for ERβ (DPN, 1 mg/kg) or ERα (PPT, 1 mg/kg) daily for the first four days of life, and then assessed adult male reproductive behavior and attractiveness via a partner preference paradigm. DPN had a greater adverse impact than PPT on reproductive behavior, suggesting a functional role for ERβ in the organization of these male-specific behaviors. Therefore the impact of neonatal ERβ agonism was further investigated by repeating the experiment using vehicle, EB and additional DPN doses (0.5 mg/kg, 1 mg/kg, and 2 mg/kg bw). Exposure to DPN suppressed male reproductive behavior and attractiveness in a dose dependent manner. Finally, males were exposed to EB or an environmentally relevant dose of genistein (GEN, 10 mg/kg), a naturally occurring xenoestrogen, which has a higher relative binding affinity for ERβ than ERα. Sexual performance was impaired by GEN but not attractiveness. In addition to suppressing reproductive behavior and attractiveness, EB exposure significantly lowered the testis to body weight ratio, and circulating testosterone levels. DPN and GEN exposure only impaired behavior, suggesting that disrupted androgen secretion does not underlie the impairment.