Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.     

Differentiation of mesenchymal cells derived from human amniotic membranes into hepatocyte-like cells in vitro

Mesenchymal stem cells are believed to be involved in the formation of mesenchymal tissues, including bone, cartilage, muscle, tendon and adipose tissue. Interestingly, it has previously been reported that mesenchymal stem cells could also differentiate into endoderm-derived cells, such as hepatocytes. The amniotic membrane contains mesenchymal cells and is a readily available human tissue. Therefore, we investigated the potential of mesenchymal cells derived from human amniotic membrane (MC-HAM) to differentiate into hepatocytes. We analyzed the expression of hepatocyte-specific genes in MC-HAM before and after induction of differentiation into hepatocytes. We observed the expression of mRNAs encoding albumin, a-fetoprotein, cytokeratin 18 and alpha1-antitrypsin, but not those encoding glucose-6-phosphatase or ornithine transcarbamylase, prior to the induction of differentiation. However, immunocytochemistry revealed that albumin and alpha-fetoprotein were abundantly produced only after the induction of differentiation into hepatocytes. In addition, we observed the storage of glycogen, a characteristic feature of hepatocytes, using periodic acid-Schiff staining of MC-HAM induced to differentiate into hepatocytes. Overall, MC-HAM appear to be able to differentiate into cells possessing some characteristics of hepatocytes. Although further studies should be carried out to determine whether such in vitro-differentiated cells can function in vivo as hepatocytes. These cells may be useful in various applications that require human hepatocytes.     

Mesenchymal stem cells: a promising candidate in regenerative medicine

Mesenchymal stem cells were initially characterized as plastic adherent, fibroblastoid cells. In recent years, there has been an increasing focus on mesenchymal stem cells since they have great plasticity and are potential for therapeutic applications. Mesenchymal stem cells or mesenchymal stem cell-like cells have been shown to reside within the connective tissues of most organs. These cells can differentiate into osteogenic, adipogenic and chondrogenic lineages under appropriate conditions. A number of reports have also indicated that these cells possess the capacity to trans-differentiate into epithelial cells and lineages derived from the neuro-ectoderm, and in addition, mesenchymal stem cells can migrate to the sites of injury, inflammation, and to tumors. These properties of mesenchymal stem cells make them promising candidates for use in regenerative medicine and may also serve as efficient delivery vehicles in site-specific therapy.     

Dose dependent effect of C-type natriuretic peptide signaling in glycosaminoglycan synthesis during TGF-β1 induced chondrogenic differentiation of mesenchymal stem cells

Recent investigations credited important roles to C-type natriuretic peptide (CNP) signaling during chondrogenesis. This study investigated the putative role of CNP in transforming growth factor (TGF)-β1 induced in vitro chondrogenic differentiation of mesenchymal stem cells (MSCs) in pellet culture. MSCs were derived from human trabecular bone and were characterized on the basis of their cell surface antigens and adipogenic, osteogenic, and chondrogenic differentiation potential. TGF-β1 induced chondrogenic differentiation and glycosaminoglycan (GAG) synthesis was analyzed on the basis of basic histology, collagen type II, Sox 9 and aggrecan expressions, and Alcian blue staining. Results revealed that human trabecular bone-derived MSCs express CNP and NPR-B analyzed on the basis of RT-PCR and immunohistochemistry. In pellet cultures of MSCs TGF-β1 successfully induced chondrogenic differentiation and GAG synthesis. RT-PCR analyses of both CNP and NPR-B during this process revealed an activation of this signaling pathway in response to TGF-β1. Similar cultures induced with TGF-β1 and treated with different doses of CNP showed that CNP supplementation at 10^?8 and 10^?7 M concentrations significantly increased GAG synthesis in a dose dependent manner, whereas at 10^?6 M concentration this stimulatory effect was diminished. In conclusion, CNP/NPR-B signaling pathway is activated during TGF-β1 induced chondrogenic differentiation of human trabecular bone-derived MSCs and may strongly be involved in GAG synthesis during this process. This effect is likely to be a dose-dependent effect.     

脂肪间充质干细胞的分离培养、多向分化及其应用前景

脂肪组织几乎遍布于动物体全身,在整个生命过程中有极强的可塑性. 近年研究表明,运用相似的分离方法,可从人、小鼠、大鼠、兔和猪等物种脂肪组织中分离获得脂肪间充质干细胞. 与骨髓来源的间充质干细胞相比,它具有相似的表面标记和分化潜能;在合适的诱导条件下,这种细胞能分别向3个胚层的细胞分化,如成肌细胞、心肌细胞、软骨细胞、成骨细胞、脂肪细胞、神经细胞、血管内皮细胞和肝细胞等;脂肪间充质干细胞具有来源丰富,取材安全方便和扩增速率高的特点,使其在细胞治疗和组织工程方面具有更广阔的应用前景.     

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.     

A novel approach for myocardial regeneration with educated cord blood cells cocultured with cells from brown adipose tissue

Umbilical cord blood (CB) is a promising source for regeneration therapy in humans. Recently, it was shown that CB was a source of mesenchymal stem cells as well as hematopoietic stem cells, and further that the mesenchymal stem cells could differentiate into a number of cells types of mesenchymal lineage, such as cardiomyocytes (CMs), osteocytes, chondrocytes, and fat cells. Previously, we reported that brown adipose tissue derived cells (BATDCs) differentiated into CMs and these CMs could adapt functionally to repair regions of myocardial infarction. In this study, we examined whether CB mononuclear cells (CBMNCs) could effectively differentiate into CMs by coculturing them with BATDCs and determined which population among CBMNCs differentiated into CMs. The results show that BATDCs effectively induced CBMNCs that were non-hematopoietic stem cells (HSCs) (educated CB cells: e-CBCs) into CMs in vitro. E-CBCs reconstituted infarcted myocardium more effectively than non-educated CBMNCs or CD34-positive HSCs. Moreover, we found that e-CBCs after 3 days coculturing with BATDCs induced the most effective regeneration for impaired CMs. This suggests that e-CBCs have a high potential to differentiate into CMs and that adequate timing of transplantation supports a high efficiency for CM regeneration. This strategy might be a promising therapy for human cardiac disease.     

Differentiation of Human Umbilical Cord Mesenchymal Stem Cells into Prostate-Like Epithelial Cells In Vivo

Although human umbilical cord mesenchymal stem cells (hUC-MSCs) have been identified as a new source of MSCs for potential application in regenerative medicine, their full potential of differentiation has not been determined. In particular, whether they have the capability to differentiate into epithelial cells of endodermal origin such as the prostate epithelial cells is unknown. Here we report that when hUC-MSCs were combined with rat urogenital sinus stromal cells (rUGSSs) and transplanted into the renal capsule in vivo, they could differentiate into prostate epithelial-like cells that could be verified by prostate epithelial cell-specific markers including the prostate specific antigen. The prostatic glandular structures formed in vivo displayed similar cellular architecture with lumens and branching features as seen for a normal prostate. In addition, the human origin of the hUC-MSCs was confirmed by immunocytochemistry for human nuclear antigen. These findings together indicate that hUC-MSCs have the capability to differentiate into epithelial-like cells that are normally derived from the endoderm, implicating their potential applications in tissue repair and regeneration of many endoderm-derived internal organs.     

Hydroxyapatite from Cuttlefish Bone: Isolation,Characterizations, and Applications

Hydroxyapatite (HA), a bioceramic, is a widely utilized material for bone tissue repair and regeneration because of its excellent properties such as biocompatibility, exceptional mechanical strength, and osteoconductivity. HA can be obtained by both synthetic and natural means. Animal bones are often considered a promising natural resource for the preparation of pure HA for biological and biomedical applications. Cuttlefish bone, also called as cuttlebone, mainly consists of calcium carbonate, and pure HA can be produced by adding phosphoric acid or ammonium hydrogen phosphate to it. Recently, cuttlefish bone-derived HA has shown promising results in terms of bone tissue repair and regeneration. The synthesized cuttlefish bone-derived has shown excellent biocompatibility, cell proliferation, increased alkaline phosphate activity, and efficient biomineralization ability with mesenchymal stem cells and osteoblastic cells. To further improve the biological properties of cuttlefish bone-derived HA, bioglass, polycaprolactone, and polyvinyl alcohol were added to it, which gave better results in terms of cell proliferation and osteogenic differentiation. Cuttlefish bone-derived HA with polymeric substances provides excellent bone formation under in vivo conditions. The studies indicate that cuttlefish bone-derived HA, along with polymeric and, protein materials, will be promising biomaterials in the field of bone tissue regeneration.     

Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep

Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.     

The use of mesenchymal stem cells in tissue engineering: A global assessment

Mesenchymal stem cells (MSCs) are of great interest to both clinicians and researchers for their great potential to enhance tissue engineering. Their ease of isolation, manipulability and potential for differentiation are specifically what have made them so attractive. These multipotent cells have been found to differentiate into cartilage, bone, fat, muscle, tendon, skin, hematopoietic-supporting stroma and neural tissue. Their diverse in vivo distribution includes bone marrow, adipose, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, blood, trabecular bone, human umbilical cord, lung, dental pulp and periodontal ligament. Despite their frequent use in research, no standardized criteria exist for the identification of mesenchymal stem cells; The International Society for Cellular Therapy has sought to change this with a set of guidelines elucidating the major surface markers found on these cells. While many studies have shown MSCs to be just as effective as unipotent cells for certain types of tissue regeneration, limitations do exist due to their immunosuppressive properties. This paper serves as a review pertaining to these issues, as well as others related to the use of MSCs in tissue engineering.Key words: mesenchymal stem cells, tissue engineering, regenerative medicine     

Stem cells from umbilical cord blood differentiate into myotubes and express dystrophin in vitro only after exposure to in vivo muscle environment

BACKGROUND INFORMATION: Duchenne muscular dystrophy is a disease characterized by progressive and irreversible muscle degeneration for which there is no therapy. HUCB (human umbilical cord blood) has been considered as an important source of haematopoietic and mesenchymal stem cells, each having been shown to differentiate into distinct cell types. However, it remains unclear if these cells are able to differentiate into muscle cells. RESULTS: We have showed that stem cells from HUCB did not differentiate into myotubes or express dystrophin when cultured in muscle-conditioned medium or with human muscle cells. However, delivery of GFP (green fluorescent protein)-transduced mononucleated cells from HUCB, which comprises both haematopoietic and mesenchymal populations, into quadriceps muscle of mdx (mouse dystrophy X-chromosome linked) mice resulted in the expression of human myogenic markers. After recovery of these cells from mdx muscle and in vitro cultivation, they were able to fuse and form GFP-positive myotubes that expressed dystrophin. CONCLUSIONS: These results indicate that chemical factors and cell-to-cell contact provided by in vitro conditions were not enough to trigger the differentiation of stem cells into muscle cells. Nevertheless, we showed that the HUCB-derived stem cells were capable of acquiring a muscle phenotype after exposure to an in vivo muscle environment, which was required to activate the differentiation programme.     

Human hair follicle-derived mesenchymal stem cells: Isolation,expansion, and differentiation

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.     

In vitro cardiomyogenic potential of human umbilical vein-derived mesenchymal stem cells

Cardiomyocyte loss in the ischemically injured human heart often leads to irreversible defects in cardiac function. Recently, cellular cardiomyoplasty with mesenchymal stem cells, which are multipotent cells with the ability to differentiate into specialized cells under appropriate stimuli, has emerged as a new approach for repairing damaged myocardium. In the present study, the potential of human umbilical cord-derived mesenchymal stem cells to differentiate into cells with characteristics of cardiomyocyte was investigated. Mesenchymal stem cells were isolated from endothelial/subendothelial layers of the human umbilical cords using a method similar to that of human umbilical vein endothelial cell isolation. Isolated cells were characterized by transdifferentiation ability to adipocytes and osteoblasts, and also with flow cytometry analysis. After treatment with 5-azacytidine, the human umbilical cord-derived mesenchymal stem cells were morphologically transformed into cardiomyocyte-like cells and expressed cardiac differentiation markers. During the differentiation, cells were monitored by a phase contrast microscope and their morphological changes were demonstrated. Immunostaining of the differentiated cells for sarcomeric myosin (MF20), desmin, cardiac troponin I, and sarcomeric alpha-actinin was positive. RT-PCR analysis showed that these differentiated cells express cardiac-specific genes. Transmission electron microscopy revealed a cardiomyocyte-like ultrastructure and typical sarcomers. These observations confirm that human umbilical cord-derived mesenchymal stem cells can be chemically transformed into cardiomyocytes and can be considered as a source of cells for cellular cardiomyoplasty.     

The use of mesenchymal stem cells in tissue engineering

Mesenchymal stem cells (MSCs) are of great interest to both clinicians and researchers for their great potential to enhance tissue engineering. Their ease of isolation, manipulability, and potential for differentiation are specifically what have made them so attractive. These multipotent cells have been found to differentiate into cartilage, bone, fat, muscle, tendon, skin, hematopoietic-supporting stroma and neural tissue. Their diverse in vivo distribution includes bone marrow, adipose, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, blood, trabecular bone, human umbilical cord, lung, dental pulp, and periodontal ligament. Despite their frequent use in research, no standardized criteria exist for the identification of mesenchymal stem cells; The International Society for Cellular Therapy has sought to change this with a set of guidelines elucidating the major surface markers found on these cells. While many studies have shown MSCs to be just as effective as unipotent cells for certain types of tissue regeneration, limitations do exist due to their immunosuppressive properties. This paper serves as a review pertaining to these issues, as well as others related to the use of MSCs in tissue engineering.     

Successful immortalization of mesenchymal progenitor cells derived from human placenta and the differentiation abilities of immortalized cells

We reported previously that mesenchymal progenitor cells derived from chorionic villi of the human placenta could differentiate into osteoblasts, adipocytes, and chondrocytes under proper induction conditions and that these cells should be useful for allogeneic regenerative medicine, including cartilage tissue engineering. However, similar to human mesenchymal stem cells (hMSCs), though these placental cells can be isolated easily, they are difficult to study in detail because of their limited life span in vitro. To overcome this problem, we attempted to prolong the life span of human placenta-derived mesenchymal cells (hPDMCs) by modifying hTERT and Bmi-1, and investigated whether these modified hPDMCs retained their differentiation capability and multipotency. Our results indicated that the combination of hTERT and Bmi-1 was highly efficient in prolonging the life span of hPDMCs with differentiation capability to osteogenic, adipogenic, and chondrogenic cells in vitro. Clonal cell lines with directional differentiation ability were established from the immortalized parental hPDMC/hTERT+Bmi-1. Interestingly, hPDMC/Bmi-1 showed extended proliferation after long-term growth arrest and telomerase was activated in the immortal hPDMC/Bmi-1 cells. However, the differentiation potential was lost in these cells. This study reports a method to extend the life span of hPDMCs with hTERT and Bmi-1 that should become a useful tool for the study of mesenchymal stem cells.     

In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract

Recent reports have suggested that mesenchymal cells derived from bone marrow may differentiate into not only mesenchymal lineage cells but also other lineage cells. There is possibility for insulin-producing cells (IPCs) to be differentiated from mesenchymal cells. We used self-functional repair stimuli of stem cells by partial injury. Rat pancreatic extract (RPE) from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to rat mesenchymal cells. After the treatment of RPE, they made clusters like islet of Langerhans within a week and expressed four pancreatic endocrine hormones; insulin, glucagon, pancreatic polypeptide, and somatostatin. Moreover, IPCs released insulin in response to normal glucose challenge. Here we demonstrate that the treatment of RPE can differentiate rat mesenchymal cells into IPCs which can be a potential source for the therapy of diabetes.     

Induced in vitro differentiation of neural-like cells from human amnion-derived fibroblast-like cells

There is growing evidence that the human amnion contains various types of stem cell. As amniotic tissue is readily available, it has the potential to be an important source of material for regenerative medicine. In this study, we evaluated the potential of human amnion-derived fibroblast-like (HADFIL) cells to differentiate into neural cells. Two HADFIL cell populations, derived from two different neonates, were analyzed. The expression of neural cell-specific genes was examined before and after in vitro induction of cellular differentiation. We found that neuron specific enolase, neurofilament-medium, beta-tubulin isotype III, and glial fibrillary acidic protein (GFAP) showed significantly increased expression following the induction of differentiation. In addition, immunostaining demonstrated that neuron specific enolase, GFAP and myelin basic protein (MBP) were present in HADFIL cells following the induction of differentiation, although one of the HADFIL cell populations showed a lower expression of GFAP and MBP. These results indicate that HADFIL cell populations have the potential to differentiate into neural cells. Although further studies are necessary to determine whether such in vitro-differentiated cells can function in vivo as neural cells, these amniotic cell populations might be of value in therapeutic applications that require human neural cells.     

Prospects for the therapeutic development of umbilical cord blood-derived mesenchymal stem cells

Umbilical cord blood (UCB) is a primitive and abundant source of mesenchymal stem cells (MSCs). UCB-derived MSCs have a broad and efficient therapeutic capacity to treat various diseases and disorders. Despite the high latent self-renewal and differentiation capacity of these cells, the safety, efficacy, and yield of MSCs expanded for ex vivo clinical applications remains a concern. However, immunomodulatory effects have emerged in various disease models, exhibiting specific mechanisms of action, such as cell migration and homing, angiogenesis, anti-apoptosis, proliferation, anti-cancer, anti-fibrosis, anti-inflammation and tissue regeneration. Herein, we review the current literature pertaining to the UCB-derived MSC application as potential treatment strategies, and discuss the concerns regarding the safety and mass production issues in future applications.