Therapeutic potential of adult bone marrow‐derived mesenchymal stem cells in diseases of the skeleton

Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late 1960s and early 1970s, MSCs or MSC‐like cells have been obtained from various mesodermal and non‐mesodermal tissues, although majority of the therapeutic applications involved bone marrow‐derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis, respectively, and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long‐term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone‐related diseases. J. Cell. Biochem. 111: 249–257, 2010. © 2010 Wiley‐Liss, Inc.     

Application of mesenchymal stem cells in regenerative medicine: A new approach in modern medical science

Mesenchymal Stem Cells (MSCs) are non-hematopoietic and multipotent stem cells, which have been considered in regenerative medicine. These cells are easily separated from different sources, such as bone marrow (BM), umbilical cord (UC), adipose tissue (AT), and etc. MSCs have the differentiation capability into chondrocytes, osteocytes, and adipocytes; This differentiation potential along with the paracrine properties have made them a key choice for tissue repair. MSCs also have various advantages over other stem cells, which is why they have been extensively studied in recent years. The effectiveness of MSCs-based therapies depend on several factors, including differentiation status at the time of use, concentration per injection, delivery method, the used vehicle, and the nature and extent of the damage. Although, MSCs have emerged promising sources for regenerative medicine, there are potential risks regarding their safety in their clinical use, including tumorigenesis, lack of availability, aging, and sensitivity to toxic environments. In this study, we aimed to discuss how MSCs may be useful in treating defects and diseases. To this aim, we will review recent advances of MSCs action mechanisms in regenerative medicine, as well as the most recent clinical trials. We will also have a brief overview of MSCs resources, differences between their sources, culture conditions, extraction methods, and clinical application of MSCs in various fields of regenerative medicine.     

Regulation of stemness and stem cell niche of mesenchymal stem cells: Implications in tumorigenesis and metastasis

Human mesenchymal stem cells (MSCs) derived from adult tissues have been considered a candidate cell type for cell‐based tissue engineering and regenerative medicine. These multipotent cells have the ability to differentiate along several mesenchymal lineages and possibly along non‐mesenchymal lineages. MSCs possess considerable immunosuppressive properties that can influence the surrounding tissue positively during regeneration, but perhaps negatively towards the pathogenesis of cancer and metastasis. The balance between the naïve stem state and differentiation is highly dependent on the stem cell niche. Identification of stem cell niche components has helped to elucidate the mechanisms of stem cell maintenance and differentiation. Ultimately, the fate of stem cells is dictated by their microenvironment. In this review, we describe the identification and characterization of bone marrow‐derived MSCs, the properties of the bone marrow stem cell niche, and the possibility and likelihood of MSC involvement in cancer progression and metastasis. J. Cell. Physiol. 222: 268–277, 2010. © 2009 Wiley‐Liss, Inc.     

Inducible Expression of Neurotrophic Factors by Mesenchymal Progenitor Cells Derived from Traumatically Injured Human Muscle

Peripheral nerve damage frequently accompanies musculoskeletal trauma and repair of these nerves could be enhanced by the targeted application of neurotrophic factors (NTFs), which are typically expressed by endogenous cells that support nerve regeneration. Injured muscle tissues express NTFs to promote reinnervation as the tissue regenerates, but the source of these factors from within the muscles is not fully understood. We have previously identified a population of mesenchymal progenitor cells (MPCs) in traumatized muscle tissue with properties that support tissue regeneration, and our hypothesis was that MPCs also secrete the NTFs that are associated with muscle tissue reinnervation. We determined that MPCs express genes associated with neurogenic function and measured the protein-level expression of specific NTFs with known functions to support nerve regeneration. We also demonstrated the effectiveness of a neurotrophic induction protocol to enhance the expression of the NTFs, which suggests that the expression of these factors may be modulated by the cellular environment. Finally, neurotrophic induction affected the expression of cell surface markers and proliferation rate of the MPCs. Our findings indicate that traumatized muscle-derived MPCs may be useful as a therapeutic cell type to enhance peripheral nerve regeneration following musculoskeletal injury.     

Impact of Aging on the Regenerative Properties of Bone Marrow-, Muscle-, and Adipose-Derived Mesenchymal Stem/Stromal Cells

Mesenchymal stem/stromal cells (MSCs) are promising cell sources for regenerative therapies due to their multipotency and ready availability, but their application can be complicated by patient-specific factors like age or illness. MSCs have been investigated for the treatment of many musculoskeletal disorders, including osteoarthritis and osteoporosis. Due to the prevalence of these diseases in older populations, researchers have studied how aging affects MSC properties and have found that proliferation and differentiation potential are impaired. However, these effects have never been compared among MSCs isolated from multiple tissue sources in the same, healthy donor. Revealing differences in how MSCs are affected by age could help identify an optimal cell source for musculoskeletal therapies targeting older patients. MSCs were isolated from young and old rabbit bone marrow, muscle, and adipose tissue. Cell yield and viability were quantified after isolation procedures, and expansion properties were assessed using assays for proliferation, senescence, and colony formation. Multipotency was also examined using lineage-specific stains and spectrophotometry of metabolites. Results were compared between age groups and among MSC sources. Results showed that MSCs are differentially influenced by aging, with bone marrow-derived stem cells having impaired proliferation, senescence, and chondrogenic response, whereas muscle-derived stem cells and adipose-derived stem cells exhibited no negative effects. While age reduced overall cell yield and adipogenic potential of all MSC populations, osteogenesis and clonogenicity remained unchanged. These findings indicate the importance of age as a factor when designing cell-based therapies for older patients.     

Mesenchymal stromal cells; a new horizon in regenerative medicine

In recent decades, mesenchymal stromal cells (MSCs) biomedical utilizing has attracted worldwide growing attention. After the first report of the human MSCs obtaining from the bone marrow (BM) tissue, these cells were isolated from wide types of the other tissues, ranging from adipose tissue to dental pulp. Their specific characteristics, comprising self-renewality, multipotency, and availability accompanied by their immunomodulatory properties and little ethical concern denote their importance in the context of regenerative medicine. Considering preclinical studies, MSCs can modify immune reactions during tissue repair and restoration, providing suitable milieu for tissue recovery; on the other hand, they can be differentiated into comprehensive types of the body cells, such as osteoblast, chondrocyte, hepatocyte, cardiomyocyte, fibroblast, and neural cells. Though a large number of studies have investigated MSCs capacities in regenerative medicine in varied animal models, the oncogenic capability of unregulated MSCs differentiation must be more assessed to enable their application in the clinic. In the current review, we provide a brief overview of MSCs sources, isolation, and expansion as well as immunomodulatory activities. More important, we try to collect and discuss recent preclinical and clinical research and evaluate current challenges in the context of the MSC-based cell therapy for regenerative medicine.     

Mesenchymal stem cells: characteristics and clinical applications

Mesenchymal stem cells (MSCs) are bone marrow populating cells, different from hematopoietic stem cells, which possess an extensive proliferative potential and ability to differentiate into various cell types, including: osteocytes, adipocytes, chondrocytes, myocytes, cardiomyocytes and neurons. MSCs play a key role in the maintenance of bone marrow homeostasis and regulate the maturation of both hematopoietic and non-hematopoietic cells. The cells are characterized by the expression of numerous surface antigens, but none of them appears to be exclusively expressed on MSCs. Apart from bone marrow, MSCs are located in other tissues, like: adipose tissue, peripheral blood, cord blood, liver and fetal tissues. MSCs have been shown to be powerful tools in gene therapies, and can be effectively transduced with viral vectors containing a therapeutic gene, as well as with cDNA for specific proteins, expression of which is desired in a patient. Due to such characteristics, the number of clinical trials based on the use of MSCs increase. These cells have been successfully employed in graft versus host disease (GvHD) treatment, heart regeneration after infarct, cartilage and bone repair, skin wounds healing, neuronal regeneration and many others. Of special importance is their use in the treatment of osteogenesis imperfecta (OI), which appeared to be the only reasonable therapeutic strategy. MSCs seem to represent a future powerful tool in regenerative medicine, therefore they are particularly important in medical research.     

Proteomic techniques for characterisation of mesenchymal stem cell secretome

Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics.     

Adult mesenchymal stem cells for tissue engineering versus regenerative medicine

Adult mesenchymal stem cells (MSCs) can be isolated from bone marrow or marrow aspirates and because they are culture-dish adherent, they can be expanded in culture while maintaining their multipotency. The MSCs have been used in preclinical models for tissue engineering of bone, cartilage, muscle, marrow stroma, tendon, fat, and other connective tissues. These tissue-engineered materials show considerable promise for use in rebuilding damaged or diseased mesenchymal tissues. Unanticipated is the realization that the MSCs secrete a large spectrum of bioactive molecules. These molecules are immunosuppressive, especially for T-cells and, thus, allogeneic MSCs can be considered for therapeutic use. In this context, the secreted bioactive molecules provide a regenerative microenvironment for a variety of injured adult tissues to limit the area of damage and to mount a self-regulated regenerative response. This regenerative microenvironment is referred to as trophic activity and, therefore, MSCs appear to be valuable mediators for tissue repair and regeneration. The natural titers of MSCs that are drawn to sites of tissue injury can be augmented by allogeneic MSCs delivered via the bloodstream. Indeed, human clinical trials are now under way to use allogeneic MSCs for treatment of myocardial infarcts, graft-versus-host disease, Crohn's Disease, cartilage and meniscus repair, stroke, and spinal cord injury. This review summarizes the biological basis for the in vivo functioning of MSCs through development and aging.     

Perinatal stem cells: A promising cell resource for tissue engineering of craniofacial bone

In facing the mounting clinical challenge and suboptimal techniques of craniofacial bone defects resulting from various conditions, such as congenital malformations, osteomyelitis, trauma and tumor resection, the ongoing research of regenerative medicine using stem cells and concurrent advancement in biotechnology have shifted the focus from surgical reconstruction to a novel stem cell-based tissue engineering strategy for customized and functional craniofacial bone regeneration. Given the unique ontogenetical and cell biological properties of perinatal stem cells, emerging evidence has suggested these extraembryonic tissue-derived stem cells to be a promising cell source for extensive use in regenerative medicine and tissue engineering. In this review, we summarize the current achievements and obstacles in stem cell-based craniofacial bone regeneration and subsequently we address the characteristics of various types of perinatal stem cells and their novel application in tissue engineering of craniofacial bone. We propose the promising feasibility and scope of perinatal stem cell-based craniofacial bone tissue engineering for future clinical application.     

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.     

The efficiency of in vitro isolation and myogenic differentiation of MSCs derived from adipose connective tissue,bone marrow,and skeletal muscle tissue

The objective of the study is to evaluate efficiency of in vitro isolation and myogenic differentiation of mesenchymal stem cells (MSCs) derived from adipose connective tissue (AD-MSCs), bone marrow (BM-MSCs), and skeletal muscle tissue (MC-MSCs). MSCs were isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue of two adult 6-wk-old rats. Cultured MSCs were treated with 5-azacytidine (AZA) to induce myogenic differentiation. Isolated MSCs and differentiated cells were evaluated by immunocytochemistry (ICC), fluorescence-activated cell sorting (FACS), PCR, and RT-PCR. AD-MSCs showed the highest proliferation rate while BM-MSCs had the lowest one. In ICC, isolated MSCs had strong CD90- and CD44-positive expression and negative expression of CD45, CD31, and CD34, while AZA-treated MSCs had strong positive desmin expression. In FACS analysis, AD-MSCs had the highest percentage of CD90- and CD44-positive-expressing cells (99% and 96%) followed by BM-MSCs (97% and 94%) and MC-MSCs (92% and 91%).At 1 wk after incubation with AZA treatment, the peak of myogenin expression reached 93% in differentiated MC-MSCs, 83.3% in BM-MSCs, and 77% in AD-MSCs. MSCs isolated from adipose connective tissue, bone marrow, and skeletal muscle tissue have the same morphology and phenotype, but AD-MSCs were the most easily accessible and had the highest rate of growth on cultivation and the highest percentage of stem cell marker expression. Moreover, although MC-MSCs showed the highest rate of myogenic differentiation potential and expression of myoblast markers, AD-MSCs and BM-MSCs still can be valuable alternatives. The differentiated myoblastic cells could be an available new choice for myoblastic auto-transplantation in regeneration medicine.     

Pooled human serum: A new culture supplement for bioreactor-based cell therapies. Preliminary results

Background

Bone Marrow MSCs are an appealing source for several cell-based therapies. Many bioreactors, as the Quantum Cell Expansion System, have been developed to generate a large number of MSCs under Good Manufacturing Practice conditions by using Human Platelet Lysate (HPL). Previously we isolated in the human bone marrow a novel cell population, named Mesodermal Progenitor Cells (MPCs), which we identified as precursors of MSCs. MPCs could represent an important cell source for regenerative medicine applications. As HPL gives rise to a homogeneus MSC population, limiting the harvesting of other cell types, in this study we investigated the efficacy of pooled human AB serum (ABS) to provide clinically relevant numbers of both MSCs and MPCs for regenerative medicine applications by using the Quantum System.

Novel aspects of parenchymal–mesenchymal interactions: from cell types to molecules and beyond

Mesenchymal stem or stromal cells (MSCs) were initially isolated from the bone marrow and received their name on the basis of their ability to differentiate into multiple lineages such as bone, cartilage, fat and muscle. However, more recent studies suggest that MSCs residing in perivascular compartments of the small and large blood vessels play a regulatory function supporting physiologic and pathologic responses of parenchymal cells, which define the functional representation of an organ or tissue. MSCs secrete or express factors that reach neighbouring parenchymal cells via either a paracrine effect or a direct cell‐to‐cell interaction promoting functional activity, survival and proliferation of the parenchymal cells. Previous concept of ‘epithelial–stromal’ interactions can now be widened. Given that MSC can also support hematopoietic, neuronal and other non‐epithelial parenchymal lineages, terms ‘parenchymal–stromal’ or ‘parenchymal–mesenchymal’ interactions may better describe the supportive or ‘trophic’ functions of MSC. Importantly, in many cases, MSCs specifically provide supportive microenvironment for the most primitive stem or progenitor populations and therefore can play a role as ‘stem/progenitor niche’ forming cells. So far, regulatory roles of MSCs have been reported in many tissues. In this review article, we summarize the latest studies that focused on the supportive function of MSC. This thread of research leads to a new perspective on the interactions between parenchymal and mesenchymal cells and justifies a principally novel approach for regenerative medicine based on co‐application of MSC and parenchymal cell for the most efficient tissue repair. Copyright © 2013 John Wiley & Sons, Ltd.     

Effect of high glucose on extensive culturing of mesenchymal stem cells derived from subcutaneous fat,omentum fat and bone marrow

Frontline research progresses the applicability of bone marrow and adipose tissue in regenerative medicine, but fails to account for the functional improvement of the diseased. The justification for the failure in terms of stem cell survival, proliferation and regeneration is unclear. However, hyperglycemia rising during pathological conditions might be one such stumbling block. The prevailing literature accounts for both detrimental and beneficial effect of high glucose on mesenchymal stem cells (MSCs) leading to perplexity. Thus, this study focuses on the effect of high glucose on mesenchymal stem cells derived from subcutaneous fat, omentum fat and bone marrow in extensive cultures. We provide evidence for the retention of MSC characteristics of all sources with regards to surface marker profiling, proliferation, differentiation and karyotyping when cultured extensively under DMEM‐HG containing glucose concentration of 25 mmol.l^–1. Thus, it can be concluded that hyperglycemia in vivo (11 mmol.l^–1) might not be a barrier for the ineffective functional improvement of transplanted stem cells. Furthermore, we elucidated subcutaneous and omentum fat as better sources of MSCs when compared with bone marrow, thereby making these sources optimal for therapies during hyperglycemic conditions. However, further research is needed to clear the path for efficient stem cell transplantation. Copyright © 2012 John Wiley & Sons, Ltd.     

Fibrin glue as the cell-delivery vehicle for mesenchymal stromal cells in regenerative medicine

The use of tissue-engineering techniques such as stem-cell therapy to renew injured tissues is a promising strategy in regenerative medicine. As a cell-delivery vehicle, fibrin glues (FG) facilitate cell attachment, growth and differentiation and, ultimately, tissue formation and organization by its three-dimensional structure. Numerous studies have provided evidence that stromal cells derived from bone marrow (bone marrow stromal cells; BMSC) and adipose tissue (adipose-derived stromal cells; ADSC) contain a population of adult multipotent mesenchymal stromal cells (MSC) and endothelial progenitor cells that can differentiate into several lineages. By combining MSC with FG, the implantation could take advantage of the mutual benefits. Researchers and physicians have pinned their hopes on stem cells for developing novel approaches in regenerative medicine. This review focuses on the therapeutic potential of MSC with FG in bone defect reconstruction, cartilage and tendon injury repair, ligament, heart and nerve regeneration, and, furthermore, wound healing.     

Characterization of in vitro cultured bone marrow and adipose tissue‐derived mesenchymal stem cells and their ability to express neurotrophic factors

MSCs (mesenchymal stem cells) have attracted attention as a promising tool for regenerative medicine and transplantation therapy. MSCs exert neuroprotective effects by secreting a number of factors in vitro and in vivo. Similar characteristics are found in ADSCs (adipose‐derived stem cells) and BMSCs (bone marrow stromal cells). Multipotent capability, easy accessibility and rapid proliferation of ADSCs have been established. Our main objective was to compare cell viability, growth rate, expression of neurotrophic factors and nestin genes in ADSCs and BMSCs. Cell doubling time and proliferation rate indicate that ADSCs has a higher proliferation rate than BMSCs. ADSCs and BMSCs express a similar pattern of CD71 and CD90 markers. Nestin immunostaining showed that ADSCs and BMSCs are immunopositive. The expression of neurotrophic factors genes in ADSCs proved similar to that of BMSCs genes. Thus adipose tissue stem cells with a high proliferation rate can express nestin and neurotrophic factor genes. Therefore ADSCs may be useful in future cell replacement therapies and help improve neurodegenerative diseases.     

Mesenchymal stromal cells from amniotic fluid are less prone to senescence compared to those obtained from bone marrow: An in vitro study

Mesenchymal stromal cells (MSCs) are considered to be an excellent source in regenerative medicine. They contain several cell subtypes, including multipotent stem cells. MSCs are of particular interest as they are currently being tested using cell and gene therapies for a number of human diseases. They represent a rare population in tissues; for this reason, they require, before being transplanted, an in vitro amplification. This process may induce replicative senescence, thus affecting differentiation and proliferative capacities. Increasing evidence suggests that MSCs from fetal tissues are significantly more plastic and grow faster than MSCs from bone marrow. Here, we compare amniotic fluid mesenchymal stromal cells (AF‐MSCs) and bone marrow mesenchymal stromal cells (BM‐MSCs) in terms of cell proliferation, surface markers, multidifferentiation potential, senescence, and DNA repair capacity. Our study shows that AF‐MSCs are less prone to senescence with respect to BM‐MSCs. Moreover, both cell models activate the same repair system after DNA damage, but AF‐MSCs are able to return to the basal condition more efficiently with respect to BM‐MSCs. Indeed, AF‐MSCs are better able to cope with genotoxic stress that may occur either during in vitro cultivation or following transplantation in patients. Our findings suggest that AF‐MSCs may represent a valid alternative to BM‐MSCs in regenerative medicine, and, of great relevance, the investigation of the mechanisms involved in DNA repair capacity of both AF‐MSCs and BM‐MSCs may pave the way to their rational use in the medical field.