The enhanced performance of bone allografts using osteogenic-differentiated adipose-derived mesenchymal stem cells

Adipose tissue was only recently considered as a potential source of mesenchymal stem cells (MSCs) for bone tissue engineering. To improve the osteogenicity of acellular bone allografts, adipose MSCs (AMSCs) and bone marrow MSCs (BM-MSCs) at nondifferentiated and osteogenic-differentiated stages were investigated in vitro and in vivo. In vitro experiments demonstrated a superiority of AMSCs for proliferation (6.1±2.3 days vs. 9.0±1.9 days between each passage for BM-MSCs, respectively, P<0.001). A significantly higher T-cell depletion (revealed by mixed lymphocyte reaction, [MLR]) was found for AMSCs (vs. BM-MSCs) at both non- and differentiated stages. Although nondifferentiated AMSCs secreted a higher amount of vascular endothelial growth factor [VEGF] in vitro (between 24 and 72 h of incubation at 0.1-21% O(2)) than BM-MSCs (P<0.001), the osteogenic differentiation induced a significantly higher VEGF release by BM-MSCs at each condition (P<0.001). After implantation in the paraspinal muscles of nude rats, a significantly higher angiogenesis (histomorphometry for vessel development (P<0.005) and VEGF expression (P<0.001)) and osteogenesis (as revealed by osteocalcin expression (P<0.001) and micro-CT imagery for newly formed bone tissue (P<0.05)) were found for osteogenic-differentiated AMSCs in comparison to BM-MSCs after 30 days of implantation. Osteogenic-differentiated AMSCs are the best candidate to improve the angio-/osteogenicity of decellularized bone allografts.     

Bone-marrow-derived mesenchymal stem cells as a target for cytomegalovirus infection: implications for hematopoiesis, self-renewal and differentiation potential

Mesenchymal stem cells (MSCs) in bone marrow (BM) regulate the differentiation and proliferation of adjacent hematopoietic precursor cells and contribute to the regeneration of mesenchymal tissues, including bone, cartilage, fat and connective tissue. BM is an important site for the pathogenesis of human cytomegalovirus (HCMV) where the virus establishes latency in hematopoietic progenitors and can transmit after reactivation to neighboring cells. Here we demonstrate that BM-MSCs are permissive to productive HCMV infection, and that HCMV alters the function of MSCs: (i) by changing the repertoire of cell surface molecules in BM-MSCs, HCMV modifies the pattern of interaction between BM-MSCs and hematopoietic cells; (ii) HCMV infection of BM-MSCs undergoing adipogenic or osteogenic differentiation impaired the process of differentiation. Our results suggest that by altering BM-MSC biology, HCMV may contribute to the development of various diseases.     

Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue

Mesenchymal stem cells (MSCs) isolated from bone marrow (BM), cartilage, and adipose tissue (AT) possess the capacity for self-renewal and the potential for multilineage differentiation, and are therefore perceived as attractive sources of stem cells for cell therapy. However, MSCs from these different sources have different characteristics. We compared MSCs of adult Sprague Dawley rats derived from these three sources in terms of their immunophenotypic characterization, proliferation capacity, differentiation ability, expression of angiogenic cytokines, and anti-apoptotic ability. According to growth curve, cell cycle, and telomerase activity analyses, MSCs derived from adipose tissue (AT-MSCs) possess the highest proliferation potential, followed by MSCs derived from BM and cartilage (BM-MSCs and C-MSCs). In terms of multilineage differentiation, MSCs from all three sources displayed osteogenic, adipogenic, and chondrogenic differentiation potential. The result of realtime RT-PCR indicated that these cells all expressed angiogenic cytokines, with some differences in expression level. Flow cytometry and MTT analysis showed that C-MSCs possess the highest resistance toward hydrogen peroxide -induced apoptosis, while AT-MSCs exhibited high tolerance to serum deprivation-induced apoptosis. Both AT and cartilage are attractive alternatives to BM as sources for isolating MSCs, but these differences must be considered when choosing a stem cell source for clinical application.     

Comparative study of the biological characteristics of mesenchymal stem cells from bone marrow and peripheral blood of rats

Mesenchymal stem cells (MSCs) from adult exhibit self-renewal and multilineage differentiation capacities, making the MSCs promising candidates for cell therapy and tissue engineering. Although bone marrow (BM) is the most universal source of MSCs, other tissues may also contain MSCs. Peripheral blood (PB), in particular, arises as the most attractive source of MSCs due to easy accessibility and noninvasive procedure. However, it is not certain that PB-MSCs have the equal biological characteristics to those of BM-MSCs. The purpose of this study was to compare the biological characteristics between BM-MSCs and PB-MSCs. We adopted granulocyte colony-stimulating factor combined with CXCR4 antagonist AMD3100 to stimulate MSCs to release into blood circulation of the rats. PB-MSCs were obtained from mobilized PB and expanded in long-term culture. BM-MSCs were isolated from the femur and tibia medullary canal of the same rats by density gradient centrifugation. After cell expansion in vitro, cell surface markers and multipotentiality analysis were performed to identify MSCs. Apoptosis resistance to H(2)O(2)-induced apoptosis, proliferation kinetics, cellular senescence, and karyotype analysis were measured to compare the biological characteristics of PB-MSCs and BM-MSCs. PB-MSCs with the typical adherent fibroblast-like morphology were similar to that of BM-MSCs. Both PB-MSCs and BM-MSCs were positive for CD44 and CD90, and negative for CD34 and CD45. They both exhibited trilineage differentiation potential and expressed lineage-specific genes. Although the BM-MSCs showed stronger osteogenic and adipogenic differentiation, PB-MSCs displayed a more chondrogenic capacity. Further, BM-MSCs have greater proliferation ability. Apoptosis resistance and cellular senescence were similar in MSCs derived from both sources. The results of our study demonstrate that PB-MSCs have similar biological characteristics to those of BM-MSCs despite certain minor differences, suggesting PB as a possible alternative source for MSCs.     

A modular approach to the engineering of a centimeter-sized bone tissue construct with human amniotic mesenchymal stem cells-laden microcarriers

Tissue engineering of clinical-relevant large tissue constructs remains a big challenge due to the mass transfer limit. A modular approach via the assembling of modular tissues thus eliminating the mass transfer limit holds great promise for fabricating centimeter-sized constructs. In the present study, we investigated the feasibility of using microcarriers seeded with adult mesenchymal stem cells (MSCs) to fabricate a large bone tissue. It was demonstrated that human amniotic MSCs (hAMSCs) were efficiently seeded onto CultiSpher S microcarriers (made of porcine gelatin) in a spinner flask and quickly proliferated while retaining a great viability. Within a total culture period of 28 days, using a two-stage culture strategy, hAMSCs-laden microcarriers with a high cell density were prepared at the first stage and the cells were then directly induced to undergo osteogenic differentiation in the same culture flask. During this cultivation process, the aggregation of cell-laden microcarriers was apparent, which resulted in aggregates of 700-800?μm, a size permissive for maintaining high cell viability. The osteogenic differentiation of hAMSCs on microcarriers was confirmed with increased mineral deposition (Alizarin red S staining and quantification of calcium content), ALP activity as well as gene expression of osteogenic markers (collagen type I and osteocalcin). These modular bone-like tissues were used as building blocks to fabricate a macroscopic bone construct in a cylindrical perfusion culture chamber (2?cm in diameter). After a 7-day perfusion culture, these modular tissues readily assembled into a centimeter-sized construct (diameter?×?height: 2?cm?×?1?cm). Both good cell viability and fairly homogenous distribution of cellular content and bone-characteristic ECM within the macrotissue were elaborated. This paper provided a proof-of-concept study for modularly engineering clinical-relevant large tissue replacements with cell-laden microcarriers.     

Role of SIRT1 and AMPK in mesenchymal stem cells differentiation

The differentiation capabilities of mesenchymal stem cells (MSCs) compromise with age and with in vitro passages which could impair the efficacy of cell therapy and tissue engineering. However, how to maintain these capabilities is not fully understood. Calorie restriction (CR, decreasing caloric intake by 30–40%) could extend longevity and reduce aging-related diseases. Recent studies revealed that CR could influence the lineage determination of stem cells including MSCs. Two important mediators of CR might be silent mating type information regulation 2 homolog 1 (SIRT1), a NAD⁺-dependent deacetylase, and AMP-activated protein kinase (AMPK), an energy-sensing kinase. Evidences are mounting that both SIRT1 and AMPK play important roles in cell fate determination of MSCs. Herein, we intend to sum up our understanding about the role of SIRT1 and AMPK in osteogenic and adipogenic potential of MSCs. Metabolic process of MSCs differentiation and the putative interplay of SIRT1 and AMPK in this process was also discussed.     

5-Aminoimidazole-4-carboxyamide ribonucleoside induces G(1)/S arrest and Nanog downregulation via p53 and enhances erythroid differentiation

Molecular mechanisms of how energy metabolism affects embryonic stem cell (ESC) pluripotency remain unclear. AMP-activated protein kinase (AMPK), a key regulator for controlling energy metabolism, is activated in response to ATP-exhausting stress. We investigated whether cellular energy homeostasis is associated with maintenance of self-renewal and pluripotency in mouse ESCs (mESCs) by using 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) as an activator of AMPK. We demonstrate that AICAR treatment activates the p53/p21 pathway and markedly inhibits proliferation of R1 mESCs by inducing G(1) /S-phase cell cycle arrest, without influencing apoptosis. Treatment with AICAR also significantly reduces pluripotent stem cell markers, Nanog and stage-specific embryonic antigen-1, in the presence of leukemia inhibitory factor, without affecting expression of Oct4. H9 human ESCs also responded to AICAR with induction of p53 activation and repression of Nanog expression. AICAR reduced Nanog mRNA levels in mESCs transiently, an effect not due to expression of miR-134 which can suppress Nanog expression. AICAR induced Nanog degradation, an effect inhibited by MG132, a proteasome inhibitor. Although AICAR reduced embryoid body formation from mESCs, it increased expression levels of erythroid cell lineage markers (Ter119, GATA1, Klf1, Hbb-b, and Hbb-bh1). Although erythroid differentiation was enhanced by AICAR, endothelial lineage populations were remarkably reduced in AICAR-treated cells. Our results suggest that energy metabolism regulated by AMPK activity may control the balance of self-renewal and differentiation of ESCs.     

FGFR2 Mutation Confers a Less Drastic Gain of Function in Mesenchymal Stem Cells Than in Fibroblasts

Gain-of-function mutations in FGFR2 cause Apert syndrome (AS), a disease characterized by craniosynostosis and limb bone defects both due to abnormalities in bone differentiation and remodeling. Although the periosteum is an important cell source for bone remodeling, its role in craniosynostosis remains poorly characterized. We hypothesized that periosteal mesenchymal stem cells (MSCs) and fibroblasts from AS patients have abnormal cell phenotypes that contribute to the recurrent fusion of the coronal sutures. MSCs and fibroblasts were obtained from the periostea of 3 AS patients (S252W) and 3 control individuals (WT). We evaluated the proliferation, migration, and osteogenic differentiation of these cells. Interestingly, S252W mutation had opposite effects on different cell types: S252W MSCs proliferated less than WT MSCs, while S252W fibroblasts proliferated more than WT fibroblasts. Under restrictive media conditions, only S252W fibroblasts showed enhanced migration. The presence of S252W mutation increased in vitro and in vivo osteogenic differentiation in both studied cell types, though the difference compared to WT cells was more pronounced in S252W fibroblasts. This osteogenic differentiation was reversed through inhibition of JNK. We demonstrated that S252W fibroblasts can induce osteogenic differentiation in periosteal MSCs but not in MSCs from another tissue. MSCs and fibroblasts responded differently to the pathogenic effects of the FGFR2(S252W) mutation. We propose that cells from the periosteum have a more important role in the premature fusion of cranial sutures than previously thought and that molecules in JNK pathway are strong candidates for the treatment of AS patients.     

Effects of a bone-like mineral film on phenotype of adult human mesenchymal stem cells in vitro

Multipotent cell types are rapidly becoming key components in a variety of tissue engineering schemes, and mesenchymal stem cells (MSCs) are emerging as an important tool in bone tissue regeneration. Although several soluble signals influencing osteogenic differentiation of MSCs in vitro are well-characterized, relatively little is known about the influence of substrate signals. This study was aimed at elucidating the effects of a bone-like mineral (BLM), which is vital in the process of bone bonding to orthopedic implant materials, on the osteogenic differentiation of human MSCs in vitro. Growth of a BLM film (carbonate apatite, Ca/P = 1.55) on poly(lactide-co-glycolide) (PLG) substrates was achieved via surface hydrolysis and subsequent incubation in a modified simulated body fluid. The BLM film demonstrated significantly increased adsorption of fibronectin, and supported enhanced proliferation of human mesenchymal stem cells (hMSCs) relative to PLG substrates. In the absence of osteogenic supplements hMSCs did not display a high expression of osteogenic markers on BLM or PLG. In the presence of osteogenic supplements hMSCs exhibited greater expression of osteogenic markers on PLG substrates than on BLM substrates, as measured by alkaline phosphatase activity and osteocalcin production. Taken together, these data support the concept that substrate signals significantly influence MSC growth and differentiation, highlighting the importance of carrier material composition in stem cell-based tissue engineering schemes.     

The effect of culture conditions on the adipogenic and osteogenic inductions of mesenchymal stem cells on micropatterned surfaces

Herein we examined an adipogenic or osteogenic induction of rat bone marrow mesenchymal stem cells (MSCs) in the corresponding media and a co-induction in a 1:1 mixed medium. The cell size effect and cell-cell contact effect were employed as two demonstrations to check the similarity or difference of the effects under these two induction ways. We seeded cells on a micropatterned surface with cell-adhesive microislands on poly(ethylene glycol) hydrogels. MSCs were well localized on the microislands separated by the strong and persistent non-fouling background, which enabled the observations of individual cells of varied sizes and numbers. We made statistics of adipogenic and osteogenic differentiations of single MSCs of different sizes (170-5600 μm(2)) and also of cell clusters of different aggregation numbers (1, 2, 4 and 8 etc.) with small, medium or large cell sizes. Both sole induction and co-induction led to monotonic cell size effects: small cells favored the adipogenic differentiation, and large cells preferred to the osteogenic differentiation. The effects of cell-cell contact were, however, rather complicated: the aggregation among cells was beneficial for both adipogenic and osteogenic differentiations, as revealed from the sole inductions; but under the co-induction culture in the mixed medium, the inherent enhancement of differentiation by the cell-cell contact encountered competition between the adipogenic and osteogenic commitments. We also examined the effects of cell density, which involved both size and contact effects, and thus exhibited different behaviors under sole induction and co-induction as well. We revealed that the density effect reflected the cooperation (for adipogenic differentiation) or competition (for osteogenic differentiation) between cell size and cell-cell contact effects.     

Comparing hydroxyapatite with osteogenic medium for the osteogenic differentiation of mesenchymal stem cells on PHBV nanofibrous scaffolds

Having advantageous biocompatibility and osteoconductive properties known to enhance the osteogenic differentiation of mesenchymal stem cells (MSCs), hydroxyapatite (HA) is a commonly used material for bone tissue engineering. What remains unclear, however, is whether HA holds a similar potential for stimulating the osteogenic differentiation of MSCs to that of a more frequently used osteogenic-inducing medium (OIM). To that end, we used PHBV electrospun nanofibrous scaffolds to directly compare the osteogenic capacities of HA with OIM over MSCs. Through the observation of cellular morphology, the staining of osteogenic markers, and the quantitative measuring of osteogenic-related genes, as well as microRNA analyses, we not only found that HA was as capable as OIM for differentiating MSCs down an osteogenic lineage; albeit, at a significantly slower rate, but also that numerous microRNAs are involved in the osteogenic differentiation of MSCs through multiple pathways involving the inhibition of cellular proliferation and stemness, chondrogenesis and adipogenesis, and the active promotion of osteogenesis. Taken together, we have shown for the first time that PHBV electrospun nanofibrous scaffolds combined with HA have a similar osteogenic-inducing potential as OIM and may therefore be used as a viable replacement for OIM for alternative in vivo-mimicking bone tissue engineering applications.     

体外构建人羊膜间充质干细胞膜片及成骨分化潜能

文题释义：细胞膜片技术：该技术避免了蛋白酶的消化和外源性支架材料的应用,通过细胞外基质分泌形成膜片组织,然后将膜片用于修复组织缺损和改善器官功能。该技术保留了大量自体细胞分泌的细胞外基质,为细胞的增殖和分化提供与体内极度相似的微环境,目前该技术已经用于临床眼角膜和食管损伤的修复。人羊膜间充质干细胞：取自于废弃的胎盘,贴壁生长,具有低免疫原性和生长周期短等特点,不仅具有成体间充质干细胞的特性还具有部分胚胎间充质干细胞的特性。  摘要背景：人羊膜间充质干细胞属于成体干细胞,其来自于废弃的胎盘,来源广泛,可以无创获取,具有免疫原性低、生长周期短等特点,是组织工程种子细胞的重要来源,目前人羊膜间充质干细胞已经用于临床糖尿病的治疗。目的：探索一种简便的方法构建人羊膜间充质干细胞膜片,并研究其成骨分化潜能。方法：将第3代人羊膜间充质干细胞高密度接种于普通培养皿中,加入成膜片诱导培养基以构建人羊膜间充质干细胞膜片,通过组织学染色以及扫描电镜观察细胞膜片的特性。取第3代人羊膜间充质干细胞高密度接种于培养皿中,加入成膜片诱导培养基培养7 d,再换用成骨诱导培养基培养14 d以构建成骨诱导的人羊膜间充质干细胞膜片。通过茜素红染色、免疫组化染色、碱性磷酸酶活性、RT-PCR以及Western blot检测人羊膜间充质干细胞膜片的成骨分化潜能。结果与结论：①苏木精-伊红染色可见人羊膜间充质干细胞膜片由多层细胞累积而成,细胞分布均匀;②扫描电镜观察可见人羊膜间充质干细胞膜片呈复层结构,胞外有大量的胞外基质产生,细胞包埋于胞外基质中;③人羊膜间充质干细胞膜片成骨诱导14 d,茜素红染色后可见橘红色沉淀,免疫组化染色后细胞周围有大量Ⅰ型胶原产生;④与未诱导的人羊膜间充质干细胞膜片相比,成骨诱导14 d后人羊膜间充质干细胞膜片碱性磷酸酶活性显著升高(P < 0.01),Ⅰ型胶原、骨钙蛋白、Runt相关转录子2的mRNA和蛋白表达量显著升高(P < 0.05);⑤该实验应用一种简便、经济的方法在普通培养皿上成功构建了人羊膜间充质干细胞膜片,体外研究证实人羊膜间充质干细胞膜片具有良好的成骨分化潜能。ORCID: 0000-0003-2163-3897(邹刚)中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Modulations of 17-beta estradiol on osteogenic and adipogenic differentiations of human mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for tissue engineering and regenerative medicine applications. However, effective regulation to improve differentiation potentials of MSCs plays a critical role in promoting successful tissue formation. Because estrogen has been demonstrated to modulate tissue and organ development and differentiation, we hypothesized that adding estrogen could effectively improve the multiple differentiation potentials of human bone marrow MSCs in vitro. In the present study, 17-beta estradiol (E2) was investigated for in vitro osteogenic and adipogenic differentiations of MSCs isolated from a healthy male human donor. After MSCs were exposed to osteogenic differentiation medium supplemented with E2 at different concentrations, osteocalcin expression is upregulated and calcium deposition (21.0%) is significantly improved ( p < 0.01; n = 4). Under adipogenic stimulation, E2 increased 35.4% lipid accumulations more than that of the group without the E2 supplement ( p < 0.01; n = 4). Estrogen's effect on osteogenesis occurs via estrogen receptors (ER)-alpha and -beta, whereas the effect on adipogenesis is through ER-alpha. Estrogen's regulation of differentiations of MSCs is dose dependent. The present study indicated that estrogen could potentially improve the role of MSCs in tissue engineering and regeneration by serving as a modulator of differentiation.     

猪的脂肪和骨髓来源间充质干细胞培养特性比较

目的明确小型猪的脂肪来源间充质干细胞（A_r_MSCs）和猪的骨髓来源间充质干细胞（BM—MSCsl体外培养特性的异同。方法广西巴马小型猪,雌雄不限,猪龄4～6个月,体质量20～30kg。AT-MSCs来源于小型猪腹股沟皮下组织．BM—MSCs来源于小型猪的骨髓组织。培养AT-MSCs和BM—MSCs并观察它们的细胞形态。流式细胞仪检测Arr_MSCs和BM—MSCs的表面标志物（CD29、CD34、CD45、CD90）。分别观察Arr-MSCs和BM—MSCs的细胞生长分化能力;实时聚合酶链反应（PCR）检测基因表达。结果流式细胞仪检测结果表明,A．r_MSCs和BM—MSCs均表达CD29[分别为（99．06±0．30）％、（99．94±0．05）％]、CD90[分别为（97．404-0．40）％、（97．43±1J29）％1阳性,CD34、CD45阴性。AT-MSCs传代需培养5～7d,而BM—MSCs需培养7～10d。与BM—MSCs比较,AT—MSCs具有更强的生长分化能力。实时PCR检测基因表达结果显示．AT—MSCs和BM—MSCs均能分化心肌特异标志物a—skeletalactin和Troponin—I．二者差异无统计学意义．表明AT—MSCs和BM—MSCs均具备多项分化潜能．结论AT—MSCs是小型猪干细胞移植治疗的理想选择．     

中药诱导骨髓间充质干细胞的成骨分化

背景：某些中药可以诱导骨髓间充质干细胞的成骨分化。骨髓间质干细胞向成骨分化的潜能,与中药治疗骨质疏松症、骨折、骨坏死、骨缺损等骨相关疾病有着理论上的相通性。 目的：了解中药诱导骨髓间充质干细胞成骨分化的发展现状,为进一步的研究奠定基础。 方法：由第一作者检索2000-01/2010-06中国期刊全文数据库(CNKI)(http://www.cnki.net/)及Pubmed数据库(http://www.ncbi.nlm.nih.gov/PubMed)。中文检索词为“中药,骨髓间充质干细胞,成骨分化”。英文检索词为“chinese herb, mesenchymal stem cells, osteogenic differentiation”。文献检索语种限定为中文和英文,纳入中药单体、单味中药、中药复方等及其含药血清在体内或体外对人或动物骨髓间充质干细胞成骨分化作用的研究文献,排除重复研究。 结果与结论：共纳入32篇文献,有关骨髓间充质干细胞研究背景的文献2篇;有关单味中药的文献5篇,其中补肾药4篇,补气药1篇;有关中药复方的文献10篇,其中补肾方6篇,补肾活血方4篇;有关中药有效组分的文献15篇。补肾、补气及活血类中药可以诱导骨髓间充质干细胞向成骨细胞分化,但以补肾类中药为主,在一定程度上阐释了“肾主骨”理论的科学内涵,并为体外大量扩增骨髓间充质干细胞、促成骨分化及组织工程骨提供了更多的种子细胞来源。     

Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities

Mesenchymal stem cells (MSCs) have important tissue repair functions and show potent immunosuppressive capacities in vitro. Although usually isolated from the bone marrow, MSCs have been identified in other tissues, including the skin and liver. In the present study, we isolated and characterized MSCs from human heart, spleen, and perirenal adipose tissue. MSCs from these different tissue sites were similar to those derived from bone marrow in that they expressed comparable levels of the cell-surface markers CD90, CD105, CD166, and HLA class I, were negative for CD34, CD45, HLA class II, CD80, and CD86 expression, and were capable of osteogenic and adipogenic differentiation. Like bone marrow-derived MSCs, MSCs from these different tissue sources inhibited the proliferation of alloactivated peripheral blood mononuclear cells (PBMCs), giving 85%, 79%, 79%, and 81% inhibition, respectively. Also in line with bone marrow-derived MSCs they inhibited proliferative responses of PBMCs to phytohemagglutinin, a nonspecific stimulator of lymphocyte proliferation, and reduced-memory T lymphocyte responses to tetanus toxoid. The results of this study demonstrate that MSCs from various tissues have similar immunophenotypes, in vitro immunosuppressive properties, and differentiation potential.