小鼠骨髓间充质干细胞的分离、培养及鉴定

目的建立一种从小鼠骨髓中分离培养间充质干细胞（MSCs）的高效方法。方法采取贴壁细胞分离法分离和纯化小鼠骨髓间充质干细胞（mMSCs）,检测mMSCs在不同诱导条件下向成骨细胞及脂肪细胞分化能力,用流式细胞术及显微镜分别检测mMSCs纯度和形态特征。结果mMSCs贴壁生长后形态较均一,细胞形态呈成纤维细胞样,流式细胞术检测：CD45、CD11b、CD44及CD29分别为（3.34）%、（2.41）%、（98.46）%及（99.36）%。第4代mMSCs经诱导后可向成骨细胞和脂肪细胞分化。结论通过贴壁培养可以从小鼠骨髓中分离培养出高纯度mMSCs,该方法效率高,稳定性好。     

The role of Wnt/β‐catenin signaling in proliferation and regeneration of the developing basilar papilla and lateral line

Canonical Wnt/β‐catenin signaling has been implicated in multiple developmental events including the regulation of proliferation, cell fate, and differentiation. In the inner ear, Wnt/β‐catenin signaling is required from the earliest stages of otic placode specification through the formation of the mature cochlea. Within the avian inner ear, the basilar papilla (BP), many Wnt pathway components are expressed throughout development. Here, using reporter constructs for Wnt/β‐catenin signaling, we show that this pathway is active throughout the BP (E6‐E14) in both hair cells (HCs) and supporting cells. To characterize the role of Wnt/β‐catenin activity in developing HCs, we performed gain‐ and loss‐of‐function experiments in vitro and in vivo in the chick BP and zebrafish lateral line systems, respectively. Pharmacological inhibition of Wnt signaling in the BP and lateral line neuromasts during the periods of proliferation and HC differentiation resulted in reduced proliferation and decreased HC formation. Conversely, pharmacological activation of this pathway significantly increased the number of HCs in the lateral line and BP. Results demonstrated that this increase was the result of up‐regulated cell proliferation within the Sox2‐positive cells of the prosensory domains. Furthermore, Wnt/β‐catenin activation resulted in enhanced HC regeneration in the zebrafish lateral line following aminoglycoside‐induced HC loss. Combined, our data suggest that Wnt/β‐catenin signaling specifies the number of cells within the prosensory domain and subsequently the number of HCs. This ability to induce proliferation suggests that the modulation of Wnt/β‐catenin signaling could play an important role in therapeutic HC regeneration. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 438–456, 2014     

Non‐lethal proteasome inhibition activates pro‐tumorigenic pathways in multiple myeloma cells

Multiple myeloma (MM) is a haematological malignancy being characterized by clonal plasma cell proliferation in the bone marrow. Targeting the proteasome with specific inhibitors (PIs) has been proven a promising therapeutic strategy and PIs have been approved for the treatment of MM and mantle‐cell lymphoma; yet, while outcome has improved, most patients inevitably relapse. As relapse refers to MM cells that survive therapy, we sought to identify the molecular responses induced in MM cells after non‐lethal proteasome inhibition. By using bortezomib (BTZ), epoxomicin (EPOX; a carfilzomib‐like PI) and three PIs, namely Rub999, PR671A and Rub1024 that target each of the three proteasome peptidases, we found that only BTZ and EPOX are toxic in MM cells at low concentrations. Phosphoproteomic profiling after treatment of MM cells with non‐lethal (IC₁₀) doses of the PIs revealed inhibitor‐ and cell type‐specific readouts, being marked by the activation of tumorigenic STAT3 and STAT6. Consistently, cytokine/chemokine profiling revealed the increased secretion of immunosuppressive pro‐tumorigenic cytokines (IL6 and IL8), along with the inhibition of potent T cell chemoattractant chemokines (CXCL10). These findings indicate that MM cells that survive treatment with therapeutic PIs shape a pro‐tumorigenic immunosuppressive cellular and secretory bone marrow microenvironment that enables malignancy to relapse.     

Activation of canonical wnt pathway promotes differentiation of mouse bone marrow‐derived MSCs into type II alveolar epithelial cells,confers resistance to oxidative stress,and promotes their migration to injured lung tissue in vitro

The differentiation of mesenchymal stem cells (MSCs) into type II alveolar epithelial (AT II) cells in vivo and in vitro, is critical for reepithelization and recovery in acute lung injury (ALI), but the mechanisms responsible for differentiation are unclear. In the present study, we investigated the role of the canonical wnt pathway in the differentiation of mouse bone marrow‐derived MSCs (mMSCs) into AT II cells. Using a modified co‐culture system with murine lung epithelial‐12 (MLE‐12) cells and small airway growth media (SAGM) to efficiently drive mMSCs differentiation, we found that GSK 3β and β‐catenin in the canonical wnt pathway were up‐regulated during differentiation. The levels of surfactant protein (SP) C, SPB, and SPD, the specific markers of AT II cells, correspondingly increased in mMSCs when Wnt3a or LiCl was added to the co‐culture system to activate wnt/β‐catenin signaling. The expression of these factors was depressed to some extent by inhibiting the pathway with the addition of DKK 1. The differentiation rate of mMSCs also depends on their abilities to accumulate and survive in inflammatory tissue. Our results suggested that the activation of wnt/β‐catenin signaling promoted mMSCs migration towards ALI mouse‐derived lung tissue in a Transwell assay, and ameliorated the cell death and the reduction of Bcl‐2/Bax induced by H₂O₂, which simultaneously caused reduced GSK 3β and β‐catenin in mMSCs. These data supports a potential mechanism for the differentiation of mMSCs into AT II cells involving canonical wnt pathway activation, which may be significant to their application in ALI. J. Cell. Physiol. 228: 1270–1283, 2013. © 2012 Wiley Periodicals, Inc.     

Clonal mesenchymal stem cells derived from human bone marrow can differentiate into hepatocyte‐like cells in injured livers of SCID mice

There is increasing evidence that human mesenchymal stem cells (hMSCs) can be a valuable, transplantable source of hepatocytes. Most of the hMSCs preparations used in these studies were likely heterogeneous cell populations, isolated by adherence to plastic surfaces or by density gradient centrifugation. Therefore, the participation of other unknown trace cell populations cannot be rigorously discounted. Here we report the isolation and establishment of a cloned human MSC line (chMSC) from human bone marrow primary culture, through which we confirmed the hepatic differentiation capability of authentic hMSCs. chMSCs expressed markers of mesenchymal cells, but not markers of hematopoietic stem cells. In vitro, chMSCs can differentiate into either mesenchymal cells or cells exhibiting hepatocyte‐like phenotypes. When transplanted intrasplentically into carbon tetrachloride‐injured livers of SCID mice, EGFP‐tagged chMSCs engrafted into the host liver parenchyma, exhibited typical hepatocyte morphology, form a three‐dimensional architecture, and differentiate into hepatocyte‐like cells expressing human albumin and α‐1‐anti‐trypsin. By confocal microscopy, ultrafine intercellular nanotubular structures were visible between adjacent transplanted and host hepatocytes. We postulate that these structures may assist in the phenotype conversion of chMSCs, possibly by exchange of cytoplasmic components between native hepatocytes and transplanted cells. Thus, a clonal pure population of hMSCs, which can be expanded in culture, may have potential as a cellular source for substitution damaged cells in hepatic injury. J. Cell. Biochem. 108: 693–704, 2009. © 2009 Wiley‐Liss, Inc.     

Guaiacol suppresses osteoclastogenesis by blocking interactions of RANK with TRAF6 and C‐Src and inhibiting NF‐κB,MAPK and AKT pathways

Angelica sinensis (AS; Dang Gui), a traditional Chinese herb, has for centuries been used for the treatment of bone diseases, including osteoporosis and osteonecrosis. However, the effective ingredient and underlying mechanisms remain elusive. Here, we identified guaiacol as the active component of AS by two‐dimensional cell membrane chromatography/C18 column/time‐of‐flight mass spectrometry (2D CMC/C18 column/TOFMS). Guaiacol suppressed osteoclastogenesis and osteoclast function in bone marrow monocytes (BMMCs) and RAW264.7 cells in vitro in a dose‐dependent manner. Co‐immunoprecipitation indicated that guaiacol blocked RANK‐TRAF6 association and RANK‐C‐Src association. Moreover, guaiacol prevented phosphorylation of p65, p50, IκB (NF‐κB pathway), ERK, JNK, c‐fos, p38 (MAPK pathway) and Akt (AKT pathway), and reduced the expression levels of Cathepsin K, CTR, MMP‐9 and TRAP. Guaiacol also suppressed the expression of nuclear factor of activated T‐cells cytoplasmic 1(NFATc1) and the RANKL‐induced Ca²⁺ oscillation. In vivo, it ameliorated ovariectomy‐induced bone loss by suppressing excessive osteoclastogenesis. Taken together, our findings suggest that guaiacol inhibits RANKL‐induced osteoclastogenesis by blocking the interactions of RANK with TRAF6 and C‐Src, and by suppressing the NF‐κB, MAPK and AKT signalling pathways. Therefore, this compound shows therapeutic potential for osteoclastogenesis‐related bone diseases, including postmenopausal osteoporosis.     

Local delivery of tetramethylpyrazine eliminates the senescent phenotype of bone marrow mesenchymal stromal cells and creates an anti‐inflammatory and angiogenic environment in aging mice

Aging drives the accumulation of senescent cells (SnCs) including stem/progenitor cells in bone marrow, which contributes to aging‐related bone degenerative pathologies. Local elimination of SnCs has been shown as potential treatment for degenerative diseases. As LepR⁺ mesenchymal stem/progenitor cells (MSPCs) in bone marrow are the major population for forming bone/cartilage and maintaining HSCs niche, whether local elimination of senescent LepR⁺ MSPCs delays aging‐related pathologies and improves local microenvironment need to be well defined. In this study, we performed local delivery of tetramethylpyrazine (TMP) in bone marrow of aging mice, which previously showed to be used for the prevention and treatment of glucocorticoid‐induced osteoporosis (GIOP). We found the increased accumulation of senescent LepR⁺ MSPCs in bone marrow of aging mice, and TMP significantly inhibited the cell senescent phenotype via modulating Ezh2‐H3k27me3. Most importantly, local delivery of TMP improved bone marrow microenvironment and maintained bone homeostasis in aging mice by increasing metabolic and anti‐inflammatory responses, inducing H‐type vessel formation, and maintaining HSCs niche. These findings provide evidence on the mechanisms, characteristics and functions of local elimination of SnCs in bone marrow, as well as the use of TMP as a potential treatment to ameliorate human age‐related skeletal diseases and to promote healthy lifespan.     

MicroRNA‐31a‐5p from aging BMSCs links bone formation and resorption in the aged bone marrow microenvironment

The alteration of age‐related molecules in the bone marrow microenvironment is one of the driving forces in osteoporosis. These molecules inhibit bone formation and promote bone resorption by regulating osteoblastic and osteoclastic activity, contributing to age‐related bone loss. Here, we observed that the level of microRNA‐31a‐5p (miR‐31a‐5p) was significantly increased in bone marrow stromal cells (BMSCs) from aged rats, and these BMSCs demonstrated increased adipogenesis and aging phenotypes as well as decreased osteogenesis and stemness. We used the gain‐of‐function and knockdown approach to delineate the roles of miR‐31a‐5p in osteogenic differentiation by assessing the decrease of special AT‐rich sequence‐binding protein 2 (SATB2) levels and the aging of BMSCs by regulating the decline of E2F2 and recruiting senescence‐associated heterochromatin foci (SAHF). Notably, expression of miR‐31a‐5p, which promotes osteoclastogenesis and bone resorption, was markedly higher in BMSCs‐derived exosomes from aged rats compared to those from young rats, and suppression of exosomal miR‐31a‐5p inhibited the differentiation and function of osteoclasts, as shown by elevated RhoA activity. Moreover, using antagomiR‐31a‐5p, we observed that, in the bone marrow microenvironment, inhibition of miR‐31a‐5p prevented bone loss and decreased the osteoclastic activity of aged rats. Collectively, our results reveal that miR‐31a‐5p acts as a key modulator in the age‐related bone marrow microenvironment by influencing osteoblastic and osteoclastic differentiation and that it may be a potential therapeutic target for age‐related osteoporosis.     

Elucidation of the Sodium‐Storage Mechanism in Hard Carbons

Hard carbons (HCs) are the most promising candidate anode materials for emerging Na‐ion batteries (NIBs). HCs are composed of misaligned graphene sheets with plentiful nanopores and defects, imparting a complex correlation between its structure and sodium‐storage behavior. The currently debated mechanism of Na⁺‐ion insertion in HCs hinders the development of high‐performance NIBs. In this article, ingenious and reliable strategies are used to elaborate the correlation between the structure and electrochemical performance and further illuminate the sodium‐storage mechanism in HCs. First, filling sulfur into the micropores of HCs can remove the low‐voltage plateau, providing solid evidence for its association with the pore‐filling mechanism. Along with the decreased concentration of defects/heteroatoms at higher treatment temperature, the reduced sloping capacity confirms the adsorption mechanism in the sloping region. Finally, the similar sodium‐insertion behaviors of HCs with ether‐based and ester‐based electrolytes indicate that no Na⁺ ions intercalate between the graphene layers. The determined adsorption‐pore‐filling mechanism encourages the design of more efficient HC anode materials with high capacity for high‐energy NIBs.     

Vertebral Bone Marrow Fat Is Positively Associated With Visceral Fat and Inversely Associated With IGF‐1 in Obese Women

Recent studies have demonstrated an important physiologic link between bone and fat. Bone and fat cells arise from the same mesenchymal precursor cell within bone marrow, capable of differentiation into adipocytes or osteoblasts. Increased BMI appears to protect against osteoporosis. However, recent studies have suggested detrimental effects of visceral fat on bone health. Increased visceral fat may also be associated with decreased growth hormone (GH) and insulin‐like growth factor 1 (IGF‐1) levels which are important for maintenance of bone homeostasis. The purpose of our study was to assess the relationship between vertebral bone marrow fat and trabecular bone mineral density (BMD), abdominal fat depots, GH and IGF‐1 in premenopausal women with obesity. We studied 47 premenopausal women of various BMI (range: 18–41 kg/m², mean 30 ± 7 kg/m²) who underwent vertebral bone marrow fat measurement with proton magnetic resonance spectroscopy (1H‐MRS), body composition, and trabecular BMD measurement with computed tomography (CT), and GH and IGF‐1 levels. Women with high visceral fat had higher bone marrow fat than women with low visceral fat. There was a positive correlation between bone marrow fat and visceral fat, independent of BMD. There was an inverse association between vertebral bone marrow fat and trabecular BMD. Vertebral bone marrow fat was also inversely associated with IGF‐1, independent of visceral fat. Our study showed that vertebral bone marrow fat is positively associated with visceral fat and inversely associated with IGF‐1 and BMD. This suggests that the detrimental effect of visceral fat on bone health may be mediated in part by IGF‐1 as an important regulator of the fat and bone lineage.     

Skin-derived multipotent stromal cells – an archrival for mesenchymal stem cells

Progenitor stem cells have been identified, isolated and characterized in numerous tissues and organs. However, their therapeutic potential and the use of these stem cells remain elusive except for a few progenitor cells from bone marrow, umbilical cord blood, eyes and dental pulp. The use of bone marrow-derived hematopoietic stem cells (HSC) or mesenchymal stem cells (MSCs) is restricted due to their extreme invasive procedures, low differentiation potential with age and rejection. Thus, we need a clinical grade alternative to progenitor stem cells with a high potential to differentiate, na?ve and is relatively easy in in vitro propagation. In this review, we summarize cell populations of adherent and floating spheres derived from different origins of skin, or correctly foreskin, by enzymatic digestion compared with established MSCs. The morphology, phenotype, differentiation capability and immunosuppressive property of the adherent cell populations are comparable with MSCs. Serum-free cultured floating spheres have limited mesodermal but higher neurogenic differentation potential, analogous to neural crest stem cells. Both the populations confirmed their plethora potential in in vitro. Together, it may be noted that the skin-derived adherent cell populations and floating cells can be good alternative sources of progenitor cells especially in cosmetic, plastic and sports regenerative medicine.     

Effect of hydrocortisone and bacterial lipopolysaccharide on colony-stimulating activity production from mouse marrow adherent cells, spleen cells and peritoneal macrophages in vitro

The effect of hydrocortisone (HC) on colony-stimulating activity (CSA) production from mouse bone marrow adherent cells, spleen cells and peritoneal macrophages with or without bacterial lipopolysaccharide (LPS) stimulation was studied. CSA in the supernatant from bone marrow adherent cells incubated with HC was found to be five times higher than CSA from cultures without LPS stimulation. In contrast, the CSA production by spleen cells and peritoneal macrophages were significantly suppressed by HC in both LPS-stimulated and non-stimulated cultures. These studies suggest that the effect of HC on CSA production was quite different depending on the target cells.     

Isolation of osteoprogenitors from murine bone marrow by selection of CD11b negative cells

Selection of cells having the most osteogenic potential is a strategy used in bone tissue engineering. Preclinical studies using murine bone marrow cells must consider the large amount of hematopoietic cells in the adherent fraction. The aim of this study was to enrich a murine bone marrow cell population with osteoprogenitors by using a simple and reliable method. Bone marrow from C57Bl/6 mice was extracted and cells which adhered onto plastic were expanded in primary culture for 14 days. Immunolabeling of the CD11b surface antigen was performed and the CD11b⁻ cell fraction was isolated by FACS. Sorted and unsorted populations were analyzed for gene expression of osteoblast differentiation, alkaline phosphatase (AlkP) activity and matrix mineralization capacities. Selection of CD11b⁻ cells increased the number of AlkP⁺ cells from the plastic adherent fraction from 6.3% ± 0.8 to 56% ± 3.3 with a sevenfold increase in AlkP activity. mRNA analysis revealed a significant increase in the CD11b⁻ fraction for Osterix (41-fold), RANKL (17-fold), M-CSF (8-fold) and Runx-2 (8-fold). An osteogenic population was obtained with improved capacities to produce a mineralized extracellular matrix in vitro, independently of the presence of glucocorticoids in the culture medium.     

Plastic adherent stromal cells from the bone marrow of commonly used strains of inbred mice: variations in yield, growth, and differentiation

Bone marrow stroma contains a unique cell population, referred to as marrow stromal cells (MSCs), capable of differentiating along multiple mesenchymal cell lineages. A standard liquid culture system has been developed to isolate MSCs from whole marrow by their adherence to plastic wherein the cells grow as clonal populations derived from a single precursor termed the colony-forming-unit fibroblast (CFU-F). Using this liquid culture system, we demonstrate that the relative abundance of MSCs in the bone marrow of five commonly used inbred strains of mice varies as much as 10-fold, and that the cells also exhibit markedly disparate levels of alkaline phosphatase expression, an early marker of osteoblast differentiation. For each strain examined, the method of isolating MSCs by plastic adherence yields a heterogeneous cell population. These plastic adherent cells also exhibit widely varying growth kinetics between the different strains. Importantly, of three inbred strains commonly used to prepare transgenic mice that we examined, only cells derived from FVB/N marrow readily expand in culture. Further analysis of cultures derived from FVB/N marrow showed that most plastic adherent cells express CD11b and CD45, epitopes of lymphohematopoietic cells. The later consists of both pre-B-cell progenitors, granulocytic and monocytic precursors, and macrophages. However, a subpopulation of the MSCs appear to represent bona fide mesenchymal progenitors, as cells can be induced to differentiate into osteoblasts and adipocytes after exposure to dexamethasone and into myoblasts after exposure to amphotericin B. Our results point to significant strain differences in the properties of MSCs and indicate that standard methods cannot be applied to murine bone marrow to isolate relatively pure populations of MSCs.     

SD大鼠骨髓间充质干细胞分离培养及鉴定的实验研究

摘要 目的：探讨应用全骨髓贴壁法体外分离培养SD大鼠骨髓间充质干细胞（BMSCs)的可行性,研究其生物学特性,为骨组织工程提供种子细胞。方法：取SPF级5周龄健康SD大鼠2只,脱颈处死,分离双下肢股骨、胫骨,全骨髓贴壁法分离培养、纯化BMSCs;通过倒置显微镜观察原代、传代细胞生长情况、绘制生长、贴壁率曲线,研究其生物学特性;流式细胞仪检测表面标志物、诱导成成骨等方法进行鉴定。结果：应用全骨髓贴壁法可在体外分离出活性好、纯度高的BMSCs。倒置显微镜下可见原代细胞呈梭形、多角形,传代细胞形态均一呈纤维样;P3代BMSCs经流式细胞鉴定：CD44、CD90高表达,CD31、CD45低表达;定向诱导向成骨细胞分化,可见明显矿化结节。结论：证实应用全骨髓贴壁培养法体外可成功分离BMSCs,所分离培养、纯化的细胞生物学稳定,纯度高、活性好,具有多向分化潜能,能为骨组织工程、骨质疏松症和骨折不愈合疾病的研究提供种子细胞。     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.