iPS cells—Alternative pluripotent cells to embryo stem cells

<正>Since the first murine and human embryonic stem cell lines were established by Drs. Evans and Kaufman [1] and Thomson et al. [2], respectively, great progress has been make in the field of     

Neural stem cells, tumour stem cells and brain tumours: dangerous relationships?

Neural stem cells (NSC) have been implicated not only in brain development and neurogenesis but also in tumourigenesis. Brain tumour stem cells (BTSC) have been isolated from several paediatric or adult human brain tumours, however their origin is still disputed. This review discusses the normal role of NSC in the adult mammalian brain and their anatomical location. It compares the molecular characteristics and the biological behaviour of NSC/BTSC, and describes the molecular pathways involved in controlling self-renewal and maintenance of adult NSC/BTSC and brain tumour development. It also assesses the current hypotheses about the origin of BTSC and the clinical consequences.     

Differentiation profile of brain tumor stem cells： a comparative study with neural stem cells

Understanding of the differentiation profile of brain tumor stem cells （BTSCs）, the key ones among tumor cell population, through comparison with neural stem cells （NSCs） would lend insight into the origin of glioma and ultimately yield new approaches to fight this intractable disease. Here, we cultured and purified BTSCs from surgical glioma specimens and NSCs from human fetal brain tissue, and further analyzed their cellular biological behaviors, especially their differentiation property. As expected, NSCs differentiated into mature neural phenotypes. In the same differentiation condition, however, BTSCs exhibited distinguished differences. Morphologically, cells grew flattened and attached for the first week, but gradually aggregated and reformed floating tumor sphere thereafter. During the corresponding period, the expression rate of undifferentiated cell marker CD 133 and nestin in BTSCs kept decreasing, but 1 week later, they regained ascending tendency. Interestingly, the differentiated cell markers GFAP and β-tubulinlII showed an expression change inverse to that of undifferentiated cell markers. Taken together, BTSCs were revealed to possess a capacity to resist differentiation, which actually represents the malignant behaviors of glioma.     

Human chorionic-plate-derived mesenchymal stem cells and Wharton’s jelly-derived mesenchymal stem cells: a comparative analysis of their potential as placenta-derived stem cells

Placenta-derived stem cells (PDSCs) have gained interest as an alternative source of stem cells for regenerative medicine because of their potential for self-renewal and differentiation and their immunomodulatory properties. Although many studies have characterized various PDSCs biologically, the properties of the self-renewal and differentiation potential among PDSCs have not yet been directly compared. We consider the characterization of chorionic-plate-derived mesenchymal stem cells (CP-MSCs) and Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) among various PDSCs and the assessment of their differentiation potential to be important for future studies into the applicability and effectiveness of PDSCs in cell therapy. In the present study, the capacities for self-renewal and multipotent differentiation of CP-MSCs and WJ-MSC isolated from normal term placentas were compared. CP-MSCs and WJ-MSCs expressed mRNAs for the pluripotent stem cell markers Oct-4, Nanog, and Sox-2. Additionally, HLA-G for immunomodulatory effects was found to be expressed at both the mRNA and protein levels in both cell types. The CP-MSCs and WJ-MSCs also had the capacities to differentiate into cells of mesodermal (adipogenic and osteogenic) and endodermal (hepatogenic) lineages. Expression of adipogenesis-related genes was higher in CP-MSCs than in WJ-MSCs, whereas WJ-MSCs accumulated more mineralized matrix than CP-MSCs. The WJ-MSCs expressed more of CYP3A4 mRNA, a marker for mature hepatocytes, than CP-MSCs. Thus, we propose that CP-MSCs and WJ-MSCs are useful sources of cells for appropriate clinical applications in the treatment of various degenerative diseases.     

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.     

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.     

A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.     

Insulin Directly Regulates Steroidogenesis via Induction of the Orphan Nuclear Receptor DAX-1 in Testicular Leydig Cells

Testosterone level is low in insulin-resistant type 2 diabetes. Whether this is due to negative effects of high level of insulin on the testes caused by insulin resistance has not been studied in detail. In this study, we found that insulin directly binds to insulin receptors in Leydig cell membranes and activates phospho-insulin receptor-β (phospho-IR-β), phospho-IRS1, and phospho-AKT, leading to up-regulation of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) gene expression in the MA-10 mouse Leydig cell line. Insulin also inhibits cAMP-induced and liver receptor homolog-1 (LRH-1)-induced steroidogenic enzyme gene expression and steroidogenesis. In contrast, knockdown of DAX-1 reversed insulin-mediated inhibition of steroidogenesis. Whether insulin directly represses steroidogenesis through regulation of steroidogenic enzyme gene expression was assessed in insulin-injected mouse models and high fat diet-induced obesity. In insulin-injected mouse models, insulin receptor signal pathway was activated and subsequently inhibited steroidogenesis via induction of DAX-1 without significant change of luteinizing hormone or FSH levels. Likewise, the levels of steroidogenic enzyme gene expression and steroidogenesis were low, but interestingly, the level of DAX-1 was high in the testes of high fat diet-fed mice. These results represent a novel regulatory mechanism of steroidogenesis in Leydig cells. Insulin-mediated induction of DAX-1 in Leydig cells of testis may be a key regulatory step of serum sex hormone level in insulin-resistant states.     

Mixed parenchymal-stromal populations of rat adrenocortical cells support the proliferation and differentiation of steroidogenic cells

Abstract. The mechanisms which regulate adrenocortical steroidogenesis in differentiated parenchymal cells have been studied in great detail. However, the stem cells that are responsible for regeneration of the adult cortex have never been identified or isolated, and their characteristics are unknown. We have developed a tissue culture system that supports the simultaneous proliferation and differentiation of steroidogenic adrenocortical cells. Utilizing density gradient separation, a cell population composed of a mixture of stromal, endothelial and parenchymal cells (MIX) was isolated from the adult rat adrenal cortex. In primary culture, MIX populations formed high saturation density multilayers from which rounded cells emerged. These cells proliferated, contained lipid, and expressed the steroidogenic enzymes δ5,3β-hydroxysteroid dehydrogenase and cholesterol side-chain cleavage cytochrome P-450scc. After selective passaging, rounded MIX-derived cells retained their steroidogenic potential, even in the absence of trophic hormone treatment. In contrast, parenchymal cells obtained from the zonae fasciculata (FASC) and glomerulosa (GLOM) respectively, formed homogeneous monolayers in primary culture, gradually de-differentiated, and no longer responded to trophic hormone treatment after passaging. Thus, primary MIX cultures provided a microenvironment that resulted in the production of adrenocortical cells with stem cell-like qualities. These cultures provide for the first time, a system for the identification of specific inducers that are responsible for both adrenocortical cytogenesis and its associated proliferation and steroidogenic differentiation.     

Involvement of ERK phosphorylation in the prevention of ischemia-induced ovarian follicular depletion by stem cells

Ovarian failure is commonly caused by aging, autoimmune disease, menopause and cancer therapy. We used an ischemic model in the ovary to test the hypothesis that stem cells are helpful for ovarian regeneration after injury. Three treatment regimes were employed: sham-operated control, ligation plus PBS, and ligation plus immortalized human bone marrow stromal cells (stem cells) groups. After ligation-induced ischemia, stem cells or PBS were injected into rat ovaries. Then, pregnant mare serum gonadotropin was given intra-peritoneally to initiate folliculogenesis. The animals were then sacrificed. The ovary gland was weighed, and ovarian folliculogenesis, stem cell differentiation and vascular neogenesis were evaluated. In order to study improvement of folliculogenesis after ovarian ischemia, steroidogenic acute regulatory protein (StAR), p44/p42 MAPK (T-ERK1/2), and phospho-p44/ p42 MAPK (P-ERK1/2) expression were specifically evaluated. Results indicated that ovarian size was smaller and that the rate of folliculogenesis was lower in ovarian ischemic-reperfusion animals, but both recovered after stem cell treatment. The stem cells migrated into the ovary and differentiated into theca cells, granulosa cells, corona radiata cells and vascular endothelial cells. In addition, von Willebrand factor (vWF) expression was increased; 17beta-estradiol (E2), progesterone (P4), P-ERK1/2 and StAR protein expression was recovered by stem cells treatment in the ischemic ovaries. The serum LH was significantly increased in ovaries of ischemia-reperfusion animals, but the stem cell treatment restored the effects. These results suggest that stem cells might be helpful for ovarian regeneration after injuries by promoting vascular neogenesis and steroidogenesis through the MAPK pathway.     

Effects of thyroid hormones on Leydig cells in the postnatal testis

Thyroid hormones (TH) stimulate oxidative metabolism in many tissues in the body, but testis is not one of them. Therefore, in this sense, testis is not considered as a target organ for TH. However, recent findings clearly show that TH have significant functions on the testis in general, and Leydig cells in particular; this begins from the onset of their differentiation through aging. Some of these functions include triggering the Leydig stem cells to differentiate, producing increased numbers of Leydig cells during differentiation by causing proliferation of Leydig stem cells and progenitors, stimulation of the Leydig cell steroidogenic function and cellular maintenance. The mechanism of action of TH on Leydig cell differentiation is still not clear and needs to be determined in future studies. However, some information on the mechanisms of TH action on Leydig cell steroidogenesis is available. TH acutely stimulate testosterone production by the Leydig cells in vitro via stimulating the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; StAR is associated with intracellular trafficking of cholesterol into the mitochondria during steroid hormone synthesis. However, the presence and/or the types of TH receptors in Leydig cells and other cell types of the Leydig cell lineage is still to be resolved. Additionally, it has been shown that thyrotropin-releasing hormone (TRH), TRH receptor and TRH mRNA in the testis in many mammalian species are seen exclusively in Leydig cells. Although the significance of the latter observations are yet to be determined, these findings prompt whether hypothalamo-pituitary-thyroid axis and hypothalamo-pituitary-testis axis are short-looped through Leydig cells.     

Interaction of thyroid hormone and steroidogenic acute regulatory (StAR) protein in the regulation of murine Leydig cell steroidogenesis

The steroidogenic acute regulatory (StAR) protein, a novel phosphoprotein, is a crucial factor involved in intramitochondrial cholesterol transportation, the rate-limiting step in steroidogenesis. The present investigations were undertaken to elucidate involvement of thyroid hormone and StAR protein in the regulation of steroidogenesis in mouse Leydig cells. Treatment of cells with triiodothyronine (T₃) coordinately augmented the levels of StAR protein, StAR mRNA, and steroid production, and these responses were progressively dependent on expression of steroidogenic factor 1 (SF-1). With regard to steroidogenesis and StAR expression, the T₃ response requires both on-going mRNA and protein synthesis. In addition, the effects of T₃ were acutely modulated at the steroidogenic machinery and luteinizing hormone receptor (LHR) function, while these levels were suppressed following longer periods of exposure to T₃. Furthermore, the inhibition of SF-1 expression by DAX-1 markedly abolished T₃-mediated StAR expression in a time frame, which was consistent with decreased steroid biosynthesis. Specific involvement of SF-1 was further confirmed by assessing the 5′-flanking region of the mouse StAR gene, which identified a region between −254 and −110 bp that was essential for T₃ function. Importantly, it was found that the SF-1 binding site at position −135 bp of the 5′-flanking region was greatly involved in T₃-mediated reporter activity. Electrophoretic mobility shift assays (EMSA) also demonstrated involvement of SF-1 in T₃ function. The relevance of T₃-mediated LHR function was investigated in mice rendered hypo-and hyperthyroid, which accounted for up-regulation in the former and down-regulation in the latter group, respectively. These findings demonstrate a key role of thyroid hormone in maintaining mouse Leydig cell function, where thyroid hormone and StAR protein coordinately regulate steroid hormone biosynthesis.     

Epigenetics and chromatin plasticity in embryonic stem cells

The study of embryonic stem cells is in the spotlight in many laboratories that study the structure and function of chromatin and epigenetic processes. The key properties of embryonic stem cells are their capacity for self-renewal and their pluripotency. Pluripotent stem cells are able to differentiate into the cells of all three germ layers, and because of this property they represent a promising therapeutic tool in the treatment of diseases such as Parkinson’s disease and diabetes, or in the healing of lesions after heart attack. As the basic nuclear unit, chromatin is responsible for the regulation of the functional status of cells, including pluripotency and differentiation. Therefore, in this review we discuss the functional changes in chromatin during differentiation and the correlation between epigenetics events and the differentiation potential of embryonic stem cells. In particular we focus on post-translational histone modification, DNA methylation and the heterochromatin protein HP1 and its unique function in mouse and human embryonic stem cells.     

The clinical potential of stem cells

Stem cells are defined by their capacity for self-renewal and multilineage differentiation, making them uniquely situated to treat a broad spectrum of human diseases. For example, because hematopoietic stem cells can reconstitute the entire blood system, bone marrow transplantation has long been used in the clinic to treat various diseases. Similarly, the transplantation of other tissue-specific stem cells, such as stem cells isolated from epithelial and neural tissues, can treat mouse disease models and human patients in which epithelial and neural cells are damaged. An alternative to tissue-specific stem cell therapy takes advantage of embryonic stem cells, which are capable of differentiating into any tissue type. Furthermore, nuclear transfer, the transfer of a post-mitotic somatic cell nucleus into an enucleated oocyte, creates a limitless source of autologous cells that, when combined with gene therapy, can serve as a powerful therapeutic tool.