Expression of cell surface markers during self-renewal and differentiation of human adipose-derived stem cells

Human adipose-derived stem cell populations express cell surface markers such as CD105, CD73, CD146 and CD140a/PDFGRα. However, it was unclear whether these markers could discriminate subpopulations of undifferentiated cells and whether the expression of these markers is modulated during differentiation. To address this issue, we analysed the immunophenotype of cultured human multipotent adipose derived stem (hMADS) cell populations at different adipocyte differentiation steps. We found that 100% of undifferentiated cells expressed CD73 and CD105. In contrast, CD146 and CD140a/PDFGRα marked two different subpopulations of cells. CD140a/PDGFRα subpopulation was regulated by FGF2, a critical factor of human adipose-derived stem cell self-renewal. During differentiation, CD73 was maintained and marked lipid-laden cells, whereas CD105 expression was inhibited in fully differentiated cells. The percentage of CD146 and CD140a/PDFGRα-positive cells declined as soon as cells had undergone differentiation. Altogether, these data support the notion that expanded adipose-derived stem cells are heterogeneous mixtures of cells and cell surface markers studied can discriminate subpopulations.     

Ferulic acid maintains the self-renewal capacity of embryo stem cells and adipose-derived mesenchymal stem cells in high fat diet-induced obese mice

Self-renewal is required for embryo stem cells (ESCs) and adipose-derived mesenchymal stem cells (ADMSCs). This study examined the ability of ferulic acid in mouse ESCs and ADMSCs, in a high fat diet-induced mouse model. Initially, five natural compounds of ferulic acid, xanthohumol, curcumin, ascorbic acid, and quercetin were screened in ESCs using an alkaline phosphate ⁺(AP⁺) assay, as a self-renewal biomarker. A ferulic acid treatment was the highest AP⁺ staining in hop-hit screening compounds. Also a ferulic acid increased Nanog mRNA levels in ESCs. The in vivo effects of ferulic acid were next examined in an obese mouse model. C57BL/6 J male mice were fed either a high fat diet (HFD) or control diet with ferulic acid (5 g/kg diet) for 8 weeks. Ferulic acid exhibited weight loss and improved glucose homeostasis, lipid profiling, and hepatic steatosis in a HFD-induced mouse model. Next, ADMSCs (Sca-1⁺CD45⁻), a hallmark of fat stem cells, were then isolated and quantified from mouse abdominal adipose tissue. A HFD decreased the Sca-1⁺CD45⁻ cell population of ADMSCs, but HFD-induced obese mice given ferulic acid showed an increased the Sca-1⁺CD45⁻ cell population of ADMSCs. Moreover, ferulic acid enhanced NANOG mRNA levels in human ADMSCs and its related gene mRNA expression. Overall, this study suggests that ferulic acid preserves self-renewal in ESCs, and contributes to ADMSCs self-renewal and effective weight control in obesity.     

Hematopoietic stem cells: generation and self-renewal

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.     

Enhanced self-renewal of hematopoietic stem/progenitor cells mediated by the stem cell gene Sall4

We reviewed preclinical data and clinical development of MDM2 (murine double minute 2), ALK (anaplastic lymphoma kinase) and PARP (poly [ADP-ribose] polymerase) inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC). Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.     

Calpain restrains the stem cells compartment in breast cancer

CAPNS1 is essential for the stability and function of ubiquitous CAPN1 and CAPN2. Calpain modulates by proteolytic cleavage many cellular substrates and its activity is often deregulated in cancer cells, therefore calpain inhibition has been proposed as a therapeutical strategy for a number of malignancies. Here we show that CAPNS1 depletion is coupled to impairment of MCF7 and MCF10AT cell lines growth on plate and defective architecture of mammary acini derived from MCF10A cells. In soft agar CAPNS1 depletion leads to cell growth increase in MCF7, and decrease in MCF10AT cells. In both MCF7 and MCF10AT, CAPNS1 depletion leads to the enlargement of the stem cell compartment, as demonstrated by mammosphere formation assays and evaluation of stem cell markers by means of FACS and western blot analysis. Accordingly, activation of calpain by thapsigargin treatment leads to a decrease in the stem cell reservoir. The expansion of the cancer stem cell population in CAPNS1 depleted cells is coupled to a defective shift from symmetric to asymmetric division during mammosphere growth coupled to a decrease in NUMB protein level.     

精原干细胞自我更新和分化的调控

精原干细胞（spermatogonial stem cells,SSCs）是体内自然状态下惟一能将遗传信息传至子代的成体干细胞,它们能通过维持自我更新和分化的稳定从而保证雄性生命过程中精子发生的持续进行。了解SSCs自我更新和分化的调节机制有助于阐明精子发生机理,并为探究其他组织中成体干细胞增殖分化的调节机制提供依据。然而目前对于SSCs自我更新和分化的调控机制所知甚少。SSCs的更新与分化遵循特定模式,受以睾丸支持细胞为主要成分的微环境及各种内分泌因素如胶质细胞源神经营养因子（GDNF）、维生素、Ets转录因子ERM/Etv5等的调控。本文评述了SSCs更新与分化的模式以及上述因素对其更新、分化的调控,探讨了其中可能涉及的信号通路,以期为本领域及其他成体干细胞相关研究提供借鉴。     

Long-term self-renewal of postnatal muscle-derived stem cells

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.     

猪脂肪干细胞的特点及其应用

脂肪干细胞(adipose-derived stem cells,ADSCs)是从脂肪组织中分离得到的一种具有多元分化潜能的干细胞,且脂肪组织在人体内的储量丰富,取材简单。因此,人源脂肪干细胞(human adipose-derived stem cells,hADSCs)具有良好的应用前景,如干细胞治疗、再生以及药物研发等。然而,要将这些基础研究成果应用于临床,必须通过临床前的安全性、可行性和潜在的风险评估。而在实验动物中,猪与人类在解剖学、遗传学和生理学上非常相似,因此猪脂肪干细胞(porcine adiposederived stem cells,pADSCs)的相关研究对人脂肪干细胞走向临床应用具有重要的理论及实践意义。基于猪脂肪干细胞的重要作用,本文综述了猪脂肪干细胞的分离、培养、免疫表型、分化能力及应用前景。     

NPR-A regulates self-renewal and pluripotency of embryonic stem cells

Self-renewal and pluripotency of embryonic stem (ES) cells are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct4, Nanog and Sox2. The mechanism regulating these signaling cascades in ES cells is of great interest. Recently, we have demonstrated that natriuretic peptide receptor A (NPR-A), a specific receptor for atrial and brain natriuretic peptides (ANP and BNP, respectively), is expressed in pre-implantation embryos and in ES cells. Here, we examined whether NPR-A is involved in the maintenance of ES cell pluripotency. RNA interference-mediated knockdown of NPR-A resulted in phenotypic changes, indicative of differentiation, downregulation of pluripotency factors (such as Oct4, Nanog and Sox2) and upregulation of differentiation genes. NPR-A knockdown also resulted in a marked downregulation of phosphorylated Akt. Furthermore, NPR-A knockdown induced accumulation of ES cells in the G1 phase of the cell cycle. Interestingly, we found that ANP was expressed in self-renewing ES cells, whereas its level was reduced after ES cell differentiation. Treatment of ES cells with ANP upregulated the expression of Oct4, Nanog and phosphorylated Akt, and this upregulation depended on NPR-A signaling, because it was completely reversed by pretreatment with either an NPR-A antagonist or a cGMP-dependent protein kinase inhibitor. These findings provide a novel role for NPR-A in the maintenance of self-renewal and pluripotency of ES cells.     

Regulation of self-renewal in normal and cancer stem cells

Mutations can confer a selective advantage on specific cells, enabling them to go through the multistep process that leads to malignant transformation. The cancer stem cell hypothesis postulates that only a small pool of low-cycling stem-like cells is necessary and sufficient to originate and develop the disease. Normal and cancer stem cells share important functional similarities such as 'self-renewal' and differentiation potential. However, normal and cancer stem cells have different biological behaviours, mainly because of a profound deregulation of self-renewal capability in cancer stem cells. Differences in mode of division, cell-cycle properties, replicative potential and handling of DNA damage, in addition to the activation/inactivation of cancer-specific molecular pathways confer on cancer stem cells a malignant phenotype. In the last decade, much effort has been devoted to unravel the complex dynamics underlying cancer stem cell-specific characteristics. However, further studies are required to identify cancer stem cell-specific markers and targets that can help to confirm the cancer stem cell hypothesis and develop novel cancer stem cell-based therapeutic approaches.     

脂肪干细胞与间充质干细胞体外诱导分化为心肌细胞的差别

本文旨在探讨脂肪干细胞(adipose-derived stem cells, ASCs)和骨髓间充质干细胞(mesenchymal stem cells, MSCs)在组织含量、体外培养和诱导分化为心肌细胞方面的差别.ASCs从新西兰白兔皮下脂肪组织提取,MSCs从大鼠四肢长骨骨髓提取,体外培养扩增,免疫细胞学方法鉴定.采用细胞集落形成法检测组织中干细胞的含量.将不同代的干细胞用不同浓度的5-氮胞苷诱导,观察其形态变化,免疫细胞化学方法检测诱导后细胞是否转化为心肌细胞.结果显示,体外培养的ASCs呈短梭形,分布均匀,生长迅速,细胞形态单一、稳定.MSCs原代生长非常缓慢,呈簇生长,细胞纯度偏低,容易混杂其它细胞类型,传代细胞容易分化和老化.脂肪组织中ASCs含量显著高于骨髓中MSCs含量,且前者含量受年龄影响小.5-氮胞苷诱导ASCs分化为心肌细胞的有效浓度为6～9μmol/L,而MSCs在3～15μmol/L 5-氮胞苷诱导下可见心肌细胞形成.ASCs诱导分化的心肌细胞呈球形细胞团,MSCs分化的心肌细胞呈条形或棒状,其心肌细胞分化率低于ASCs.幼年动物MSCs的组织含量和心肌细胞分化率均高于老年动物,而ASCs受动物年龄影响较小.结果表明,ASCs在组织含量、细胞纯度、生长速度和心肌细胞分化率等方面均明显优于骨髓MSCs,在心肌细胞再生方面较MSCs具有更大的优势.     

Mammary epithelial stem cells: transplantation and self-renewal analysis

Abstract.  An entire mammary epithelial outgrowth, capable of full secretory differentiation, may be comprised of the progeny of a single cellular antecedent. This conclusion is based upon the maintenance of retroviral insertion sites within the somatic DNA of successive transplant generations derived from a single mammary fragment. In addition, dissociation of these clonal dominant glands and implantation of dispersed cells at limiting dilution demonstrated that both duct-limited and lobule-limited outgrowths were developed, as well as complete, fully differentiated glands. Thus, transplantation has revealed three distinct mammary epithelial progenitors in the mouse. Similar studies have extended this observation to rat mammary tissue. Recently, using cre-lox conditional activation of reporter genes, a new epithelial progenitor, specific for mammary secretory epithelium in postlactation females has been uncovered. In situ , these cells were shown to regenerate secretory lobules upon successive pregnancies. In transplant studies, they demonstrated the capacity for self-renewal and contributed to the new generation of all of the known epithelial cell types among mammary epithelium. In limiting dilution, the parity-induced progenitors were capable of engendering lobule-limited and duct-limited outgrowths in their entirety, but not completely developed glands. Serial transplant studies indicate that these progenitors have a significant but limited capacity for self-renewal.     

CrxOS maintains the self-renewal capacity of murine embryonic stem cells

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiated morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.