Clinical translation of stem cells: insight for cartilage therapies

Abstract

The limited regenerative capacity of articular cartilage and deficiencies of current treatments have motivated the investigation of new repair technologies. In vitro cartilage generation using primary cell sources is limited by cell availability and expansion potential. Pluripotent stem cells possess the capacity for chondrocytic differentiation and extended expansion, providing a potential future solution to cell-based cartilage regeneration. However, despite successes in producing cartilage using adult and embryonic stem cells, the translation of these technologies to the clinic has been severely limited. This review discusses recent advances in stem cell-based cartilage tissue engineering and the major current limitations to clinical translation of these products. Concerns regarding appropriate animal models and studies, stem cell manufacturing, and relevant regulatory processes and guidelines will be addressed. Understanding the significant hurdles limiting the clinical use of stem cell-based cartilage may guide future developments in the fields of tissue engineering and regenerative medicine.     

From gristle to chondrocyte transplantation: treatment of cartilage injuries

This review addresses the progress in cartilage repair technology over the decades with an emphasis on cartilage regeneration with cell therapy. The most abundant cartilage is the hyaline cartilage that covers the surface of our joints and, due to avascularity, this tissue is unable to repair itself. The cartilage degeneration seen in osteoarthritis causes patient suffering and is a huge burden to society. The surgical approach to cartilage repair was non-existing until the 1950s when new surgical techniques emerged. The use of cultured cells for cell therapy started as experimental studies in the 1970s that developed over the years to a clinical application in 1994 with the introduction of the autologous chondrocyte transplantation technique (ACT). The technology is now spread worldwide and has been further refined by combining arthroscopic techniques with cells cultured on matrix (MACI technology). The non-regenerating hypothesis of cartilage has been revisited and we are now able to demonstrate cell divisions and presence of stem-cell niches in the joint. Furthermore, cartilage derived from human embryonic stem cells and induced pluripotent stem cells could be the base for new broader cell treatments for cartilage injuries and the future technology base for prevention and cure of osteoarthritis.     

Emergence of chondrogenic progenitor stem cells in transplantation biology-prospects and drawbacks

Avascular tissues such as a cartilage contains a unique type of cell called as the chondrocyte. We, however, have not understood the origin of the chondrocyte population and how this population is maintained in the normal tissue. In spite of being considered to be a simple tissue, scientist had always faced difficulties to engineer this tissue. This is because different structural regions of the articular cartilage were never understood. In addition to this, the limited self-repair potential of cartilage tissue and lack of effective therapeutic options for the treatment of damaged cartilage has remained an unsolved problem. Mesenchymal stem cell based therapy may provide a solution for cartilage regeneration. This is due to their ability to differentiate into chondrogenic lineage when appropriate conditions are provided. An ideal cell source, a three-dimensional cell culture, a suitable scaffold material that accomplishes all the necessary properties and bioactive factors in specific amounts are required to induce chondrocyte differentiation and proliferation. Cartilage tissue engineering is a promising and rapidly expanding area of research that assures cartilage regeneration. However, many unsolved questions concerning the mechanism of engraftment of chondrocytes following transplantation in vivo, biological safety after transplantation and the retention of these cells for lifetime remain to be addressed that is possible only through years of extensive research. Further studies are therefore required to estimate the long-term sustainability of these cells in the native tissue, to identify well suited delivery materials and to have a thorough understanding of the mechanism of interaction between the chondrocytes and extracellular matrix and time is not far when this cell based therapy will provide a comprehensive cure to cartilage disease.     

Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: Perspectives from stem cell biology and molecular medicine

Adipose‐derived stem cells (ASCs) have been discovered for more than a decade. Due to the large numbers of cells that can be harvested with relatively little donor morbidity, they are considered to be an attractive alternative to bone marrow derived mesenchymal stem cells. Consequently, isolation and differentiation of ASCs draw great attention in the research of tissue engineering and regenerative medicine. Cartilage defects cause big therapeutic problems because of their low self‐repair capacity. Application of ASCs in cartilage regeneration gives hope to treat cartilage defects with autologous stem cells. In recent years, a lot of studies have been performed to test the possibility of using ASCs to re‐construct damaged cartilage tissue. In this article, we have reviewed the most up‐to‐date articles utilizing ASCs for cartilage regeneration in basic and translational research. Our topic covers differentiation of adipose tissue derived mesenchymal stem cells into chondrocytes, increased cartilage formation by co‐culture of ASCs with chondrocytes and enhancing chondrogenic differentiation of ASCs by gene manipulation. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Cellules souches et biomatériaux injectables pour la médecine régénératrice du cartilage : le consortium « chondrograft »

Articular cartilage is a non innerved, nonvascularized and poorly cellularized connective tissue that is frequently damaged as a result of trauma or age-linked degenerative diseases. It hardly heals spontaneously and its alterations often lead to further extracellular matrix degradation and ultimately, to the loss of joint function. Past decades, many therapeutic approaches have been developed to improve the poor intrinsic self-repair properties of cartilage. Unfortunately, these techniques have not proved really satisfying. In this context, the regeneration of a functional cartilage through tissue engineering and regenerative medicine has recently been contemplated. In particular, the transplantation of autologous reparative cells using a synthetic biomaterial appears promising. We have thus developed and patented a biocompatible self-setting cellulose hydrogel that can be used as an injectable scaffold for cell-based regenerative medicine. Our studies associate this hydrogel with adult mesenchymal stem cells derived from adipose tissue, as a source of reparative cells for cartilage tissue engineering. In a first set of experiments, we have determined the optimal culture conditions required to induce the controlled chondrogenic commitment of stem cells (morphogens, hypoxia, three-dimensional environments…). The preclinical potential of hybrid constructs associating cells and hydrogel has then been assessed with success in animals (mouse, rabbit). Today, trauma and degenerative pathologies of joint tissues remain a major challenge for clinicians and cartilage engineers. Establishing the proof of concept of hydrogel-associated stem cells-based regenerative medicine could help us open new therapeutic windows in the treatment of joint disorders.     

Isolation of human nasoseptal chondrogenic cells: a promise for cartilage engineering

In cartilaginous tissues, perichondrium cambium layer may be the source of new cartilage. Human nasal septal perichondrium is considered to be a homogeneous structure in which some authors do not recognize the perichondrium internal zone or the cambium layer as a layer distinct from adjacent cartilage surface. In the present study, we isolated a chondrogenic cell population from human nasal septal cartilage surface zone. Nasoseptal chondrogenic cells were positive for surface markers described for mesenchymal stem cells, with exception of CD146, a perivascular cell marker, which is consistent with their avascular niche in cartilage. Although only Sox-9 was constitutively expressed, they also revealed osteogenic and chondrogenic, but not adipogenic, potentials in vitro, suggesting a more restricted lineage potential compared to mesenchymal stem cells. Interestingly, even in absence of chondrogenic growth factors in the pellet culture system, nasoseptal chondrogenic cells had a capacity to synthesize sulfated glycosaminoglycans, large amounts of collagen type II and to a lesser extent collagen type I. The spontaneous chondrogenic potential of this population of cells indicates that they may be a possible source for cartilage tissue engineering. Besides, the pellet culture system using nasoseptal chondrogenic cells may also be a model for studies of chondrogenesis.     

Molecular and cellular characterization during chondrogenic differentiation of adipose tissue-derived stromal cells in vitro and cartilage formation in vivo

Human adipose tissue is a viable source of mesenchymal stem cells (MSCs) with wide differentiation potential for musculoskeletal tissue engineering research. The stem cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and expanded in vitro easily. This study was to determine molecular and cellular characterization of PLA cells during chondrogenic differentiation in vitro and cartilage formation in vivo . When cultured in vitro with chondrogenic medium as monolayers in high density, they could be induced toward the chondrogenic lineages. To determine their ability of cartilage formation in vivo , the induced cells in alginate gel were implanted in nude mice subcutaneously for up to 20 weeks. Histological and immunohistochemical analysis of the induced cells and retrieved specimens from nude mice at various intervals showed obviously cartilaginous phenotype with positive staining of specific extracellular matrix (ECM). Correlatively, results of RT-PCR and Western Blot confirmed the expression of characteristic molecules during chondrogenic differentiation namely collagen type II, SOX9, cartilage oligomeric protein (COMP) and the cartilage-specific proteoglycan aggrecan. Meanwhile, there was low level synthesis of collagen type X and decreasing production of collagen type I during induction in vitro and formation of cartilaginous tissue in vivo . These cells induced to form engineered cartilage can maintain the stable phenotype and indicate no sign of hypertrophy in 20 weeks in vivo , however, when they cultured as monolayers, they showed prehypertrophic alteration in late stage about 10 weeks after induction. Therefore, it is suggested that human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis and forming engineered cartilage.     

The potential therapeutic use of stem cells in cartilage repair

As our population demographics change, osteoarthritis and cartilage defects are becoming more prevalent. The discovery of stems cells and their ability for indefinite regeneration has revolutionised the way cartilage problems are viewed. Tissue engineering has been shown to be the ideal way of repairing articular cartilage lesions, i.e. back to native tissue. Cartilage is an ideal tissue engineering target as it is avascular, aneural and alymphatic. The two main types of stem cells being investigated in chondrogenesis are embryological and mesenchymal stem cells. Research into embryological stem cells has been surrounded by controversy because of ethical, religious and social concerns. We discuss the use of embryological and mesenchymal stem cells in cartilage repair and the various factors involved in the differentiation into chondrocytes. We also discuss commonly used mesenchymal stem cell markers and their limitations.     

Mesenchymal stem cell therapy in osteoarthritis: advanced tissue repair or intervention with smouldering synovial activation?

Although it is generally accepted that osteoarthritis is a degenerative condition of the cartilage, other tissues such as synovium in which immunological and inflammatory reactions occur contribute to the development of joint pathology. This sheds new light on the potential mechanism of action of mesenchymal stem cell therapy in osteoarthritis. Rather than tissue repair due to local transformation of injected mesenchymal stem cells to chondrocytes and filling defects in cartilage, such treatment might suppress synovial activation and indirectly ameliorate cartilage damage. Desando and co-workers report in Arthritis Research & Therapy that intra-articular delivery of adipose-derived stem cells attenuates progression of synovial activation and joint destruction in osteoarthritis in an experimental rabbit model. Clinical studies are warranted to see whether this approach might be a novel way to combat development of joint destruction in inflammatory subtypes of osteoarthritis.     

Stem cell-based approaches to solving the problem of tissue supply for islet transplantation in type 1 diabetes

Type 1 diabetes is a debilitating condition, affecting millions worldwide, that is characterized by the autoimmune destruction of insulin-producing pancreatic islets of Langerhans. Although exogenous insulin administration has traditionally been the mode of treatment for this disease, recent advancements in the transplantation of donor-derived insulin-producing cells have provided new hope for a cure. However, in order for islet transplantation to become a widely used technique, an alternative source of cells must be identified to supplement the limited supply currently available from cadaveric donor organs. Stem cells represent a promising solution to this problem, and current research is being aimed at the creation of islet-endocrine tissue from these undifferentiated cells. This review presents a summary of the research to date involving stem cells and cell replacement therapy for type 1 diabetes. The potential for the differentiation of embryonic stem (ES) cells to islet phenotype is discussed, as well as the possibility of identifying and exploiting a pancreatic progenitor/stem cell from the adult pancreas. The possibility of creating new islets from adult stem cells derived from other tissues, or directly form other terminally differentiated cell types is also addressed. Finally, a model for the isolation and maturation of islets from the neonatal porcine pancreas is discussed as evidence for the existence of an islet precursor cell in the pancreas.     

Novel insights in basic and applied stem cell therapy

The achievement of novel findings in stem cell research were the subject of the meeting organized by Stem Cell Research Italy (SCR Italy) and by the International Society for Cellular Therapy-Europe (ISCT). Stem cell therapy represents great promise for the future of molecular and regenerative medicine. The use of several types of stem cells is a real opportunity to provide a valid approach to curing several untreatable human diseases. Before it is suitable for clinical applications, stem cell biology needs to be investigated further and in greater detail. Basic stem cell research could provide exact knowledge regarding stem cell action mechanisms, and pre-clinical research on stem cells on an in vivo model of disease provides scientific evidence for future human applications. Applied stem cell research is a promising new approach to handling several diseases. Along with tissue engineering, it offers a new and promising discipline that can help to manage human pathologies through stem cell therapy. All of these themes were discussed in this meeting, covering stem cell subtypes with their newest basic and applied research.     

Cartilage tissue engineering: Molecular control of chondrocyte differentiation for proper cartilage matrix reconstruction

Background

Articular cartilage defects are a veritable therapeutic problem because therapeutic options are very scarce. Due to the poor self-regeneration capacity of cartilage, minor cartilage defects often lead to osteoarthritis. Several surgical strategies have been developed to repair damaged cartilage. Autologous chondrocyte implantation (ACI) gives encouraging results, but this cell-based therapy involves a step of chondrocyte expansion in a monolayer, which results in the loss in the differentiated phenotype. Thus, despite improvement in the quality of life for patients, reconstructed cartilage is in fact fibrocartilage. Successful ACI, according to the particular physiology of chondrocytes in vitro, requires active and phenotypically stabilized chondrocytes.

Scope of review

This review describes the unique physiology of cartilage, with the factors involved in its formation, stabilization and degradation. Then, we focus on some of the most recent advances in cell therapy and tissue engineering that open up interesting perspectives for maintaining or obtaining the chondrogenic character of cells in order to treat cartilage lesions.

Major conclusions

Current research involves the use of chondrocytes or progenitor stem cells, associated with “smart” biomaterials and growth factors. Other influential factors, such as cell sources, oxygen pressure and mechanical strain are considered, as are recent developments in gene therapy to control the chondrocyte differentiation/dedifferentiation process.

General significance

This review provides new information on the mechanisms regulating the state of differentiation of chondrocytes and the chondrogenesis of mesenchymal stem cells that will lead to the development of new restorative cell therapy approaches in humans. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

种子细胞——组织工程连载之三

种子细胞也是组织工程的核心研究内容,获得足够数量和质量的种子细胞是开展体外组织工程的必要基础。用于组织工程的种子细胞必须具有形成新组织结构的能力,主要来源于自体、同种异体或异种,在具体应用时各有利弊。一些成体干细胞由于不存在伦理争议以及发育分化条件相对简单等优势是重要的种子细胞,包括造血干细胞、骨髓干细胞、神经干细胞、脂肪干细胞、皮肤干细胞。人胚胎干细胞及其组织工程要真正在临床医学中得到应用,还有很长的一段路要走。其他一些细胞也可以作为组织工程种子细胞,包括内皮细胞、上皮细胞、成纤维细胞、骨细胞、成骨细胞、角质细胞、前脂肪细胞、脂肪细胞、肌腱细胞等。这些细胞已分化,分裂能力有限,但仍应用于组织工程。理想的种子细胞具有一定标准。     

人退变椎间盘软骨终板干细胞多向分化的研究

目的:为了分离和鉴定人退变椎间盘软骨终板干细胞。方法:收集因腰椎间盘退变性疾病行腰椎间盘摘除术并植骨融合的标本。在解剖显微镜下清理软骨终板组织,并消化软骨终板,提取软骨终板细胞。获得的软骨终板细胞经过琼脂糖三维筛选系统培养后,选取细胞克隆团并进行体外扩增,扩增后的细胞行流式细胞术检测干细胞标志物证实退变软骨终板中存在干细胞。结果:共聚焦免疫荧光提示退变椎间盘软骨终板组织中存在干细胞标志物STRO1、CD105、CD73、CD90阳性的细胞。经琼脂糖三维培养基筛选的CESCs在免疫表型上符合干细胞标准。结论:在人退变椎间盘的软骨终板中存在具有多向分化潜能的干细胞。     

Adult mesenchymal stem cells: a pluripotent population with multiple applications

Mesenchymal stem cells (MSCs) have been isolated not only from bone marrow, but also from many other tissues such as adipose tissue, skeletal muscle, liver, brain and pancreas. Because MSC were found to have the ability to differentiate into cells of multiple organs and systems such as bone, fat, cartilage, muscle, neurons, hepatocytes and insulin-producing cells, MSCs have generated a great deal of interest for their potential use in regenerative medicine and tissue engineering. Furthermore, given the ease of their isolation and their extensive expansion rate and differentiation potential, mesenchymal stem cells are among the first stem cell types that have a great potential to be introduced in the clinic. Finally, mesenchymal stem cells seem to be not only hypoimmunogenic and thus be suitable for allogeneic transplantation, but they are also able to produce immunosuppression upon transplantation. In this review we summarize the latest research in the use of mesenchymal stem cells in transplantation for generalized diseases, local implantation for local tissue defects, and as a vehicle for genes in gene therapy protocols.     

Stem cells for osteoarticular and vascular tissue engineering

Tissue damages or loss of organs often result in structural and metabolic changes that can cause serious complications. The therapeutic objective of tissue engineering (TE) is to recreate, regenerate or restore function of damaged tissue. TE is based on the coalescence of three components: a scaffold or matrix from natural or synthetic origin biodegradable or not, reparative cells and signals (hypoxia, mechanical stress, morphogens…). Articular cartilage, bone and blood vessels are tissues for which TE has progressed significantly, from basic research to clinical trials. If biomaterials must exhibit different properties depending on the tissue to regenerate, the cellular component of TE is mostly represented by stem cells notably adult mesenchymal stem cells harvested from bone marrow or adipose tissue. In recent years, progress has been made in our understanding of the biological mechanisms that govern stem cell differentiation and in the development of materials with controlled physicochemical and biological properties. However, many technological barriers and regulations concerns have to be overcome before tissue engineering enters into the therapeutic arsenal of regenerative medicine. This review aims at highlighting the progress in the use of stem cells for engineering osteoarticular and vascular tissues.     

Engineered Co-culture Strategies Using Stem Cells for Facilitated Chondrogenic Differentiation and Cartilage Repair

Osteoarthritis (OA) is a chronic disease in elders and athletes due to limited regenerative capacities of cartilage tissues and subsequently insufficient recovery of damaged sites. Recent clinical treatments for OA have utilized progenitor cell-based therapies for cartilage tissue regeneration. Administration of a single type of cell population such as stem cells or chondrocytes does not guarantee a full recovery of cartilage defects. Therefore, current tissue engineering approaches using co-culture techniques have been developed to mimic complex and dynamic cellular interactions in native cartilage tissues and facilitate changes in cellular phenotypes into chondrogenesis. Therefore, this paper introduces recently developed co-culture systems using two major cell populations, mesenchymal stem cells (MSCs) and chondrocytes. Specifically, a series of examples to describe (1) synergistic in vitro activations of MSCs by paracrine signaling molecules from adult chondrocytes in co-culture systems and (2) functional in vivo tissue regeneration via co-administration of both cell types were reviewed. Based on these findings, it could be speculated that engineered co-culture systems using MSC/ chondrocyte is a promising and feasible cell-based OA therapy in clinical aspects.     

Tissue engineering and cartilage

Human articular cartilage is an avascular structure, which, when injured, poses significant hurdles to repair strategies. Not only does the defect need to be repopulated with cells, but preferentially with hyaline-like cartilage.Successful tissue engineering relies on four specific criteria: cells, growth factors, scaffolds, and the mechanical environment. The cell population utilized may originate from cartilage itself (chondrocytes) or from growth factors that direct the development of mesenchymal stem cells toward a chondrogenic phenotype. These stem cells may originate from various mesenchymal tissues including bone marrow, synovium, adipose tissue, skeletal muscle, and periosteum. Another unique population of multipotent cells arises from Wharton''s jelly in human umbilical cords. A number of growth factors have been associated with chondrogenic differentiation of stem cells and the maintenance of the chondrogenic phenotype by chondrocytes in vitro, including TGFβ; BMP-2, 4 and 7; IGF-1; and GDF-5.Scaffolds chosen for effective tissue engineering with respect to cartilage repair can be protein based (collagen, fibrin, and gelatin), carbohydrate based (hyaluronan, agarose, alginate, PLLA/PGA, and chitosan), or formed by hydrogels. Mechanical compression, fluid-induced shear stress, and hydrostatic pressure are aspects of mechanical loading found in within the human knee joint, both during gait and at rest. Utilizing these factors may assist in stimulating the development of more robust cells for implantation.Effective tissue engineering has the potential to improve the quality of life of millions of patients and delay future medical costs related to joint arthroplasty and associated procedures.Key words: cartilage repair, gene therapy, growth factors, biomaterials, tissue engineering, stem cells, chondrocyte     

The Effect of Dexamethasone and Triiodothyronine on Terminal Differentiation of Primary Bovine Chondrocytes and Chondrogenically Differentiated Mesenchymal Stem Cells

The newly evolved field of regenerative medicine is offering solutions in the treatment of bone or cartilage loss and deficiency. Mesenchymal stem cells, as well as articular chondrocytes, are potential cells for the generation of bone or cartilage. The natural mechanism of bone formation is that of endochondral ossification, regulated, among other factors, through the hormones dexamethasone and triiodothyronine. We investigated the effects of these hormones on articular chondrocytes and chondrogenically differentiated mesenchymal stem cells, hypothesizing that these hormones would induce terminal differentiation, with chondrocytes and differentiated stem cells being similar in their response. Using a 3D-alginate cell culture model, bovine chondrocytes and chondrogenically differentiated stem cells were cultured in presence of triiodothyronine or dexamethasone, and cell proliferation and extracellular matrix production were investigated. Collagen mRNA expression was measured by real-time PCR. Col X mRNA and alkaline phosphatase were monitored as markers of terminal differentiation, a prerequisite of endochondral ossification. The alginate culture system worked well, both for the culture of chondrocytes and for the chondrogenic differentiation of mesenchymal stem cells. Dexamethasone led to an increase in glycosaminoglycan production. Triiodothyronine increased the total collagen production only in chondrocytes, where it also induced signs of terminal differentiation, increasing both collagen X mRNA and alkaline phosphatase activity. Dexamethasone induced terminal differentiation in the differentiated stem cells. The immature articular chondrocytes used in this study seem to be able to undergo terminal differentiation, pointing to their possible role in the onset of degenerative osteoarthritis, as well as their potential for a cell source in bone tissue engineering. When chondrocyte-like cells, after their differentiation, can indeed be moved on towards terminal differentiation, they can be used to generate a model of endochondral ossification, but this limitation must be kept in mind when using them in cartilage tissue engineering application.