Human bone marrow-derived mesenchymal stem cells differentiate into epidermal-like cells in vitro

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a population of pluripotent cells. They can differentiate into different embryonic layer cells as osteoblasts, adipocytes, chondrocytes, myoblasts, neurocytes, etc. However, there are only few reports with regard to differentiate hMSCs into epidermal cells in vitro. In this study, we want to investigate the feasibility of inducing hMSCs into epidermal-like cells under specific medium in vitro. hMSCs in specific inducing medium expressed the early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), the late differentiated marker, the pan-cytokeratin, and another early marker, the beta1-integrin, which up-regulated remarkably in inducing medium. Their morphologies were changed from spindle-like fibroblastic appearances to oblate or irregular shapes under phase contrast microscopy. The hemidesmosome structure was found using the transmission electron microscope. All these data suggested that, under certain conditions, hMSCs have the potential to differentiate into epidermal-like cells. It will be of great accordance in the study of the multipotential property of hMSCs.     

Effectiveness of human mesenchymal stem cells derived from bone marrow cryopreserved for 23-25 years

Objective

To evaluate long-term cryopreserved human bone marrow cells (BMCs) as a source of functional mesenchymal stem cells (MSCs).

Methods

Samples of human BMCs that were cryopreserved for 23–25 years (n = 20) were thawed to obtain an initial culture and a primary culture (P₀) that was propagated through five passages (P₁–P₅) to obtain MSCs. Freshly collected human bone marrow samples (n = 20) were used as controls for comparison of efficiency of recovery and growth characteristics of MSCs. P₃ cultures were tested for their capacity to differentiate into osteoblasts, adipocytes, and neuronal cells. Appropriate staining, immunohistochemical and biochemical methods were employed to ascertain cell type identities at different stages of culturing.

Results

In the initial culture, the cell adherence rate of the cryopreserved cells was significantly lower than that of controls (19.7% vs. 38.2%, p < 0.05) while the relative rate of recovery of MSCs was only 48.5 ± 8.6% in P₀. At the end of P₃, fibroblast-like cells accounted for about 95% of cells in both cryopreserved and control groups (p > 0.05). These cells were positive for essential MSC surface molecules (CD90, CD105, CD166, CD44, CD29, CD71, CD73) and negative for haematopoietic and endothelial cell markers (CD45, CD34, HLA-DR). The cell growth and cell cycle patterns were similar for both groups. MSCs at P₃ from both groups had similar capacities to differentiate in vitro into osteoblasts, adipocytes, and neuronal cells.

Conclusion

Using the methods described here, long-term (23–25 years) cryopreserved human BMCs can be successfully cultivated to obtain MSCs that have good differentiation capabilities.     

Phenotypic characterization and in vivo localization of human adipose-derived mesenchymal stem cells

Human adipose-derived mesenchymal stem cells (hADMSCs) are a potential cell source for autologous cell therapy due to their regenerative ability. However, detailed cytological or phenotypic characteristics of these cells are still unclear. Therefore, we determined and compared cell size, morphology, ultrastructure, and immunohistochemical (IHC) expression profiles of isolated hADMSCs and cells located in human adipose tissues. We also characterized the localization of these cells in vivo. Light microscopy examination at low power revealed that hADMSCs acquired a spindle-shaped morphology after four passages. Additionally, high power views showed that these cells had various sizes, nuclear contours, and cytoplasmic textures. To further evaluate cell morphology, transmission electron microscopy was performed. hADMSCs typically had ultrastructural characteristics similar to those of primitive mesenchymal cells including a relatively high nuclear/cytosol ratio, prominent nucleoli, immature cytoplasmic organelles, and numerous filipodia. Some cells contained various numbers of lamellar bodies and lipid droplets. IHC staining demonstrated that PDGFR and CD10 were constitutively expressed in most hADMSCs regardless of passage number but expression levels of α-SMA, CD68, Oct4 and c-kit varied. IHC staining of adipose tissue showed that cells with immunophenotypic characteristics identical to those of hADMSCs were located mainly in the perivascular adventitia not in smooth muscle area. In summary, hADMSCs were found to represent a heterogeneous cell population with primitive mesenchymal cells that were mainly found in the perivascular adventitia. Furthermore, the cell surface markers would be CD10/PDGFR. To obtain defined cell populations for therapeutic purposes, further studies will be required to establish more specific isolation methods.     

胚胎干细胞向造血干/祖细胞定向诱导分化的研究进展

胚胎干细胞(embryonic stem cell,ES细胞)是指由胚胎内细胞团(inner cell mass,ICM)细胞经体外抑制培养而筛选得到的细胞,具有无限增殖潜能,在体外可以向造血细胞分化,有可能为造血干细胞移植和血细胞输注开辟新的来源．此外,ES细胞向造血干/祖细胞的定向诱导分化也为阐明哺乳动物造血发育的细胞和分子机制提供了良好的体外模型．对ES细胞向造血干/祖细胞定向分化的研究进展进行了综述．     

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.     

Trafficking and differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are a heterogeneous population of stem/progenitor cells with pluripotent capacity to differentiate into mesodermal and non‐mesodermal cell lineages, including osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons. MSCs reside primarily in the bone marrow, but also exist in other sites such as adipose tissue, peripheral blood, cord blood, liver, and fetal tissues. When stimulated by specific signals, these cells can be released from their niche in the bone marrow into circulation and recruited to the target tissues where they undergo in situ differentiation and contribute to tissue regeneration and homeostasis. Several characteristics of MSCs, such as the potential to differentiate into multiple lineages and the ability to be expanded ex vivo while retaining their original lineage differentiation commitment, make these cells very interesting targets for potential therapeutic use in regenerative medicine and tissue engineering. The feasibility for transplantation of primary or engineered MSCs as cell‐based therapy has been demonstrated. In this review, we summarize the current knowledge on the signals that control trafficking and differentiation of MSCs. J. Cell. Biochem. 106: 984–991, 2009. © 2009 Wiley‐Liss, Inc.     

Human platelet lysate supports ex vivo expansion and enhances osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

MSCs (mesenchymal stem cells) with their versatile growth and differentiation potential are ideal candidates for use in regenerative medicine and are currently making their way into clinical trials, which requires the development of xeno-free protocols for their culture. In this study, MSCs were cultured in 10% FCS or 7.5% HPL (human platelet lysate)-supplemented media. We found that both groups of MSCs showed a comparable morphology, phenotype and proliferation. The percentage of cells in the S- and G2-/M-phases, however, was slightly up-regulated (P<0.01) in HPL group. HPL contains PDGF (platelet derived growth factor)-AB and IGF (insulin-like growth factor)-1. In addition, compared with FCS group, MSCs in HPL group showed an increase in osteogenic differentiation and a decrease in adipogenic differentiation. In conclusion, MSCs in HPL-supplemented media maintained similar growing potential and phenotype, while osteogenic potential was enhanced. HPL offers a promising alternative to FCS for MSC expansion for clinical application, especially in bone injury diseases.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Differentiation potential of bone marrow mesenchymal stem cells in duck

The bone marrow mesenchymal stem cells （MSCs） are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the me- dium. The primary MSCs were expanded for 11 passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different- passage MSCs expressed CD44, ICAM-1 and SSEA-4, but not CD34, CD45 and SSEA-1 when detected by immunofluorescence staining There was no significant difference among the positive rates of passages 2, 6 and 8 （P 〉 0.05）, but a significant difference existed among those of passages 2, 6, 8 and 11 （P 〈 0.05）. After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase （ALP）, and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 （P〉0.05）, but a significant difference existed among those of passages 3, 4, 6 and 8 （P〈0.05）. These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.     

Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells

We have previously shown that cells isolated from the outer ears of adult mice are a source of mesenchymal stem cells that can be induced to differentiate into adipo-, osteo-, and chondrocytes. In this study, we demonstrate that ear mesenchymal stem cells (EMSC) express stromal cell-associated markers (CD44, CD73) and stem cell marker Sca-1 and can be differentiated into spontaneously contracting muscle cells. Treatment of cells with epidermal growth factor (EGF) change their morphology from fibroblast shapes into stick-like structures that show repeated spontaneous contractions. Under conditions that promote myogenic differentiation, EMSC expressed mRNA for myoD and ventricular specific myosin light chain (MLC-2v) and protein for connexin 43, sarcomeric alpha-actinin, myocyte enhancer factor 2c (MEF2c), myosin heavy chain (MyHC), myogenin, and sarco-endoplasmic reticulum Ca(2+)ATPase (SERCA) 1. However, the cells were negative for Nkx2.5, GATA4, and ANP. Intracellular Ca(2+) transients in spontaneously beating EMSC, visualized by Fluo-3AM, showed a frequency of Ca(2+) oscillations ranging over 28-59/min (mean 41.17 +/- SEM 1.54). We also demonstrated that small pieces of ear tissues (ear punches) collected from live mice provide sufficient numbers of EMSC to isolate, culture and differentiate them into myocytes. Due to the ease of acquiring an expanding repertoire of differentiated EMSC cell types by a noninvasive surgical procedure, we conclude that the ear may prove to be a potential source of autologous cells for regenerative medicine, as supported by the fact that ears are one of the best sources of cells for somatic cell nuclear transfer (SCNT).     

Modeling of hemophilia A using patient-specific induced pluripotent stem cells derived from urine cells

Aims

Hemophilia A (HA) is a severe, congenital bleeding disorder caused by the deficiency of clotting factor VIII (FVIII). For years, traditional laboratory animals have been used to study HA and its therapies, although animal models may not entirely mirror the human pathophysiology. Human induced pluripotent stem cells (iPSCs) can undergo unlimited self-renewal and differentiate into all cell types. This study aims to generate hemophilia A (HA) patient-specific iPSCs that differentiate into disease-affected hepatocyte cells. These hepatocytes are potentially useful for in vitro disease modeling and provide an applicable cell source for autologous cell therapy after genetic correction.

Main methods

In this study, we mainly generated iPSCs from urine collected from HA patients with integration-free episomal vectors PEP4-EO2S-ET2K containing human genes OCT4, SOX2, SV40LT and KLF4, and differentiated these iPSCs into hepatocyte-like cells. We further identified the genetic phenotype of the FVIII genes and the FVIII activity in the patient-specific iPSC derived hepatic cells.

Key findings

HA patient-specific iPSCs (HA-iPSCs) exhibited typical pluripotent properties evident by immunostaining, in vitro assays and in vivo assays. Importantly, we showed that HA-iPSCs could differentiate into functional hepatocyte-like cells and the HA-iPSC-derived hepatocytes failed to produce FVIII, but otherwise functioned normally, recapitulating the phenotype of HA disease in vitro.

Significance

HA-iPSCs, particular those generated from the urine using a non-viral approach, provide an efficient way for modeling HA in vitro. Furthermore, HA-iPSCs and their derivatives serve as an invaluable cell source that can be used for gene and cell therapy in regenerative medicine.     

In vitro Osteogenic impulse effect of Dexamethasone on periodontal ligament stem cells

Periodontium is a complex organ composed of mineralized epithelial and connective tissue. Dexamethasone could stimulateproliferation of osteoblast and fibroblasts. This study aimed to assess the osteogenic effect of dexamethasone on periodentalligament (PDL) stem cells. PDL stem cells were collected from periodontal ligament tissue of root of extracted premolar of youngand healthy people. The stem cells were cultured in α-MEM Medium in three groups, one group with basic medium contains (α-MEM and FBS 10 % and 50 mmol of β_ gelisrophosphat and L_ ascorbic acid µg/ml), the second group: basic medium withdexamethasone and the third one: basic medium without any osteogenic stimulant. Mineralization of cellular layer was analyzedwith Alizarin red stain method. Osteogenic analysis was done by Alkaline phosphates and calcium test. These analysis indicatedthat the amount of intra-cellular calcium and alkaline phosphates in the Dexamethasone group was far more than the control andbasic group (P<0.05). The results of Alizarin red stain indicated more mineralization of cultured cells in Dexamethasone group(P<0.05). The study results showed that Dexamethasone has significant osteogenic effect on PDL stem cells and further studies arerecommended to evaluate its effect on treatment of bone disorders.     

Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources

Human mesenchymal stem cells (MSC) from adult and fetal tissues are promising candidates for cell therapy but there is a need to identify the optimal source for bone regeneration. We have previously characterized MSC populations in first trimester fetal blood, liver, and bone marrow and we now evaluate their osteogenic differentiation potential in comparison to adult bone marrow MSC. Using quantitative real-time RT-PCR, we demonstrated that 16 osteogenic-specific genes (OC, ON, BSP, OP, Col1, PCE, Met2A, OPG, PHOS1, SORT, ALP, BMP2, CBFA1, OSX, NOG, IGFII) were expressed in both fetal and adult MSC under basal conditions and were up-regulated under osteogenic conditions both in vivo and during an in vitro 21-day time-course. However, under basal conditions, fetal MSC had higher levels of osteogenic gene expression than adult MSC. Upon osteogenic differentiation, fetal MSC produced more calcium in vitro and reached higher levels of osteogenic gene up-regulation in vivo and in vitro. Second, we observed a hierarchy within fetal samples, with fetal bone marrow MSC having greater osteogenic potential than fetal blood MSC, which in turn had greater osteogenic potential than fetal liver MSC. Finally, we found that the level of gene expression under basal conditions was positively correlated with both calcium secretion and gene expression after 21 days in osteogenic conditions. Our findings suggest that stem cell therapy for bone dysplasias such as osteogenesis imperfecta may benefit from preferentially using first trimester fetal blood or bone marrow MSC over fetal liver or adult bone marrow MSC.     

人脐带间充质干细胞作为维持人胚胎干细胞生长饲养层细胞的研究

目的寻找可以维持人胚胎干细胞未分化生长的人源性细胞作为饲养层细胞,从而解决使用鼠源性细胞作为饲养层带来的安全问题。方法尝试以人脐带间充质干细胞作为饲养层细胞来培养人胚胎干细胞,检验其是否可以维持人胚胎干细胞的未分化生长状态。用胶原酶消化法分离人脐带间充质干细胞,光镜下观察细胞形态;流式细胞仪检测其表面标志;诱导人脐带间充质干细胞向成骨细胞和脂肪细胞进行分化。将人胚胎干细胞系H1接种于丝裂霉素C灭活后的人脐带间充质干细胞上,每隔5d进行一次传代。培养20代后,对人胚胎干细胞特性进行相关检测,包括细胞形态、碱性磷酸酶染色、相关多能性基因的表达、分化能力。结果从人脐带中分离出的间充质干细胞为梭形,呈平行排列生长或漩涡状生长;细胞高表达CD44、CD29、CD73、CD105、CD90、CD86、CD147、CD117,不表达CD14、CD38、CD133、CD34、CD45、HLA-DR;具有分化成脂肪细胞和成骨细胞的潜能。人胚胎干细胞在人脐带间充质干细胞饲养层上培养20代后,继续保持人胚胎干细胞的典型形态,碱性磷酸酶染色为阳性,免疫荧光染色显示OCT4、Nanog、SSEA4、TRA-1-81、TRA-1-60的表达为阳性,SSEA1表达为阴性,体外悬浮培养可以形成拟胚体。结论人脐带间充质干细胞可以作为人胚胎干细胞的饲养层细胞,支持其生长,并维持其未分化生长状态。     

Human mesenchymal stem cells in contact with their environment: surface characteristics and the integrin system

The identification of mesenchymal stem cells (MSCs) in adult human tissues and the disclosure of their self-renew-al and multi-lineage differentiation capabilities have provided exciting prospects for cell-based regeneration and tis-sue engineering. Although a considerable amount of data is available describing MSCs, there is still lack of information regarding the molecular mechanisms that govern their adhesion and migration. In this work, we will review the current state of knowledge on integrins and other adhesion molecules found to be expressed on MSCs. The discussed topics include the characteristics of MSCs and their clinical applications, integrins and their central role in cell-matrix attachment and migration, and comments on mobilization, differentiation and contribution to tumour development. Finally, by understanding the complex and fundamental pathways by which MSCs attach and migrate, it might be possible to fine-tune the strategies for effective and safe use of MSCs in regenerative therapies.