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.     

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there are many challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.     

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.     

Bridging the regeneration gap: stem cells, biomaterials and clinical translation in bone tissue engineering

Advances in our understanding of skeletal stem cells and their role in bone development and repair, offer the potential to open new frontiers in bone regeneration. Tissue engineering seeks to harness the regenerative capacity innate to bone for the replacement of tissue lost or damaged through a broad range of conditions associated with an increasingly aged population. The strategy entails ex vivo expansion of multipotential populations followed by delivery to the site of damage on dynamically durable-biodegradable three-dimensional structures which provide the requisite extracellular microenvironment for stem cell driven tissue development. This review will examine bone stem cell biology, and current advances in skeletal tissue engineering through the enhancement and marrying of biologically informed and clinically relevant strategies.     

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells

Background aimsHuman mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populationsMethodsGiven these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD modelResultsOur study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD.ConclusionsOur results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.     

Identification and clonal characterisation of a progenitor cell sub-population in normal human articular cartilage

Background

Articular cartilage displays a poor repair capacity. The aim of cell-based therapies for cartilage defects is to repair damaged joint surfaces with a functional replacement tissue. Currently, chondrocytes removed from a healthy region of the cartilage are used but they are unable to retain their phenotype in expanded culture. The resulting repair tissue is fibrocartilaginous rather than hyaline, potentially compromising long-term repair. Mesenchymal stem cells, particularly bone marrow stromal cells (BMSC), are of interest for cartilage repair due to their inherent replicative potential. However, chondrocyte differentiated BMSCs display an endochondral phenotype, that is, can terminally differentiate and form a calcified matrix, leading to failure in long-term defect repair. Here, we investigate the isolation and characterisation of a human cartilage progenitor population that is resident within permanent adult articular cartilage.

Methods and Findings

Human articular cartilage samples were digested and clonal populations isolated using a differential adhesion assay to fibronectin. Clonal cell lines were expanded in growth media to high population doublings and karyotype analysis performed. We present data to show that this cell population demonstrates a restricted differential potential during chondrogenic induction in a 3D pellet culture system. Furthermore, evidence of high telomerase activity and maintenance of telomere length, characteristic of a mesenchymal stem cell population, were observed in this clonal cell population. Lastly, as proof of principle, we carried out a pilot repair study in a goat in vivo model demonstrating the ability of goat cartilage progenitors to form a cartilage-like repair tissue in a chondral defect.

Conclusions

In conclusion, we propose that we have identified and characterised a novel cartilage progenitor population resident in human articular cartilage which will greatly benefit future cell-based cartilage repair therapies due to its ability to maintain chondrogenicity upon extensive expansion unlike full-depth chondrocytes that lose this ability at only seven population doublings.     

Human platelet lysate enhances proliferation but not chondrogenic differentiation of pediatric mesenchymal progenitors

Background aimsCell therapies have the potential to improve reconstructive procedures for congenital craniofacial cartilage anomalies such as microtia. Adipose-derived stem cells (ADSCs) and auricular cartilage stem/progenitor cells (CSPCs) are promising candidates for cartilage reconstruction, but their successful use in the clinic will require the development of xeno-free expansion and differentiation protocols that can maximize their capacity for chondrogenesis.MethodsWe assessed the behavior of human ADSCs and CSPCs grown either in qualified fetal bovine serum (FBS) or human platelet lysate (hPL), a xeno-free alternative, in conventional monolayer and 3-dimensional spheroid cultures.ResultsWe show that CSPCs and ADSCs display greater proliferation rate in hPL than FBS and express typical mesenchymal stromal cell surface antigens in both media. When expanded in hPL, both cell types, particularly CSPCs, maintain a spindle-like morphology and lower surface area over more passages than in FBS. Both media supplements support chondrogenic differentiation of CSPCs and ADSCs grown either as monolayers or spheroids. However, chondrogenesis appears less ordered in hPL than FBS, with reduced co-localization of aggrecan and collagen type II in spheroids.ConclusionshPL may be beneficial for the expansion of cells with chondrogenic potential and maintaining stemness, but not for their chondrogenic differentiation for tissue engineering or disease modeling.     

Use of human induced pluripotent stem cells for cartilage regeneration in vitro and within chondral defect models of knee joint cartilage in vivo: a Preferred Reporting Items for Systematic Reviews and Meta-Analyses systematic literature review

Background aimsArticular cartilage has limited regenerative ability when damaged through trauma or disease. Failure to treat focal chondral lesions results in changes that inevitably progress to osteoarthritis. Osteoarthritis is a major contributor to disability globally, which results in significant medical costs and lost wages every year. Human induced pluripotent stem cells (hiPSCs) have long been considered a potential autologous therapeutic option for the treatment of focal chondral lesions. Although there are significant advantages to hiPSCs over other stem cell options, such as mesenchymal and embryonic stem cells, there are concerns regarding their ability to form bona fide cartilage and their tumorgenicity in vivo.MethodsThe authors carried out a systematic literature review on the use of hiPSCs to produce differentiated progeny capable of producing high-quality cartilage in vitro and regenerate cartilage in osteochondral defects in vivo in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Eight studies were included in the review that used hiPSCs or their derived progeny in xenogeneic transplants in animal models to regenerate cartilage in osteochondral defects of the knee joint. The in vitro-differentiated, hiPSC-derived and in vivo defect repair ability of the hiPSC-derived progeny transplants were assessed.ResultsMost studies reported the generation of high-quality cartilage-producing progeny that were able to successfully repair cartilage defects in vivo. No tumorigenicity was observed.ConclusionsThe authors conclude that hiPSCs offer a valuable source of cartilage-producing progeny that show promise as an effective cell-based therapy in treating focal chondral lesions.     

Manufacture of a human mesenchymal stem cell population using an automated cell culture platform

Tissue engineering and regenerative medicine are rapidly developing fields that use cells or cell-based constructs as therapeutic products for a wide range of clinical applications. Efforts to commercialise these therapies are driving a need for capable, scaleable, manufacturing technologies to ensure therapies are able to meet regulatory requirements and are economically viable at industrial scale production. We report the first automated expansion of a human bone marrow derived mesenchymal stem cell population (hMSCs) using a fully automated cell culture platform. Differences in cell population growth profile, attributed to key methodological differences, were observed between the automated protocol and a benchmark manual protocol. However, qualitatively similar cell output, assessed by cell morphology and the expression of typical hMSC markers, was obtained from both systems. Furthermore, the critical importance of minor process variation, e.g. the effect of cell seeding density on characteristics such as population growth kinetics and cell phenotype, was observed irrespective of protocol type. This work highlights the importance of careful process design in therapeutic cell manufacture and demonstrates the potential of automated culture for future optimisation and scale up studies required for the translation of regenerative medicine products from the laboratory to the clinic.     

Stem cell-based composite tissue constructs for regenerative medicine

A major task of contemporary medicine and dentistry is restoration of human tissues and organs lost to diseases and trauma. A decade-long intense effort in tissue engineering has provided the proof of concept for cell-based replacement of a number of individual tissues such as the skin, cartilage, and bone. Recent work in stem cell-based in vivo restoration of multiple tissue phenotypes by composite tissue constructs such as osteochondral and fibro-osseous grafts has demonstrated probable clues for bioengineered replacement of complex anatomical structures consisting of multiple cell lineages such as the synovial joint condyle, tendon-bone complex, bone-ligament junction, and the periodontium. Of greater significance is a tangible contribution by current attempts to restore the structure and function of multitissue structures using cell-based composite tissue constructs to the understanding of ultimate biological restoration of complex organs such as the kidney or liver. The present review focuses on recent advances in stem cell-based composite tissue constructs and attempts to outline challenges for the manipulation of stem cells in tailored biomaterials in alignment with approaches potentially utilizable in regenerative medicine of human tissues and organs.     

Adult human neural stem cell therapeutics: Current developmental status and prospect

Over the past two decades, regenerative therapies using stem cell technologies have been developed for various neurological diseases. Although stem cell therapy is an attractive option to reverse neural tissue damage and to recover neurological deficits, it is still under development so as not to show significant treatment effects in clinical settings. In this review, we discuss the scientific and clinical basics of adult neural stem cells (aNSCs), and their current developmental status as cell therapeutics for neurological disease. Compared with other types of stem cells, aNSCs have clinical advantages, such as limited proliferation, inborn differentiation potential into functional neural cells, and no ethical issues. In spite of the merits of aNSCs, difficulties in the isolation from the normal brain, and in the in vitro expansion, have blocked preclinical and clinical study using aNSCs. However, several groups have recently developed novel techniques to isolate and expand aNSCs from normal adult brains, and showed successful applications of aNSCs to neurological diseases. With new technologies for aNSCs and their clinical strengths, previous hurdles in stem cell therapies for neurological diseases could be overcome, to realize clinically efficacious regenerative stem cell therapeutics.     

Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine

Human exfoliated deciduous teeth have been considered to be a promising source for regenerative therapy because they contain unique postnatal stem cells from human exfoliated deciduous teeth (SHED) with self-renewal capacity, multipotency and immunomodulatory function. However preservation technique of deciduous teeth has not been developed. This study aimed to evaluate that cryopreserved dental pulp tissues of human exfoliated deciduous teeth is a retrievable and practical SHED source for cell-based therapy. SHED isolated from the cryopreserved deciduous pulp tissues for over 2 years (25–30 months) (SHED-Cryo) owned similar stem cell properties including clonogenicity, self-renew, stem cell marker expression, multipotency, in vivo tissue regenerative capacity and in vitro immunomodulatory function to SHED isolated from the fresh tissues (SHED-Fresh). To examine the therapeutic efficacy of SHED-Cryo on immune diseases, SHED-Cryo were intravenously transplanted into systemic lupus erythematosus (SLE) model MRL/lpr mice. Systemic SHED-Cryo-transplantation improved SLE-like disorders including short lifespan, elevated autoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended increased interleukin 17-secreting helper T cells in MRL/lpr mice systemically and locally. SHED-Cryo-transplantation was also able to recover osteoporosis bone reduction in long bones of MRL/lpr mice. Furthermore, SHED-Cryo-mediated tissue engineering induced bone regeneration in critical calvarial bone-defect sites of immunocompromised mice. The therapeutic efficacy of SHED-Cryo transplantation on immune and skeletal disorders was similar to that of SHED-Fresh. These data suggest that cryopreservation of dental pulp tissues of deciduous teeth provide a suitable and desirable approach for stem cell-based immune therapy and tissue engineering in regenerative medicine.     

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.     

Low intensity ultrasound as a supporter of cartilage regeneration and its engineering

Ultrasound (US) is being used widely in clinic for diagnostic and therapeutic purposes, but clinical utilization of low intensity ultrasound (LIUS) has been very limited. However, therapeutic potential of LIUS has been reported in animal models of musculoskeletal system disorders, and its application is being expanded in various fields. This review will focus on the application of LIUS on the cartilage tissue engineering and repair of cartilage disorder such as osteoarthritis (OA). We will introduce our experimental results showing the LIUS effects on the chondrocyte viability, proliferation and matrix protein synthesisin vitro, and its application in the cartilage tissue engineering using mesenchymal stem cells (MSCs)in vivo. Also the current status on the issues will be discussed by comparing our results with those of other laboratories. In conclusion, we suggest that LIUS is an efficient and clinically applicable method for cartilage tissue engineering and cartilage repair.     

Mesenchymal stem cells in arthritic diseases

Mesenchymal stem cells (MSCs), the nonhematopoietic progenitor cells found in various adult tissues, are characterized by their ease of isolation and their rapid growth in vitro while maintaining their differentiation potential, allowing for extensive culture expansion to obtain large quantities suitable for therapeutic use. These properties make MSCs an ideal candidate cell type as building blocks for tissue engineering efforts to regenerate replacement tissues and repair damaged structures as encountered in various arthritic conditions. Osteoarthritis (OA) is the most common arthritic condition and, like rheumatoid arthritis (RA), presents an inflammatory environment with immunological involvement and this has been an enduring obstacle that can potentially limit the use of cartilage tissue engineering. Recent advances in our understanding of the functions of MSCs have shown that MSCs also possess potent immunosuppression and anti-inflammation effects. In addition, through secretion of various soluble factors, MSCs can influence the local tissue environment and exert protective effects with an end result of effectively stimulating regeneration in situ. This function of MSCs can be exploited for their therapeutic application in degenerative joint diseases such as RA and OA. This review surveys the advances made in the past decade which have led to our current understanding of stem cell biology as relevant to diseases of the joint. The potential involvement of MSCs in the pathophysiology of degenerative joint diseases will also be discussed. Specifically, we will explore the potential of MSC-based cell therapy of OA and RA by means of functional replacement of damaged cartilage via tissue engineering as well as their anti-inflammatory and immunosuppressive activities.     

Three-dimensional bioprinting in tissue engineering and regenerative medicine

With the advances of stem cell research, development of intelligent biomaterials and three-dimensional biofabrication strategies, highly mimicked tissue or organs can be engineered. Among all the biofabrication approaches, bioprinting based on inkjet printing technology has the promises to deliver and create biomimicked tissue with high throughput, digital control, and the capacity of single cell manipulation. Therefore, this enabling technology has great potential in regenerative medicine and translational applications. The most current advances in organ and tissue bioprinting based on the thermal inkjet printing technology are described in this review, including vasculature, muscle, cartilage, and bone. In addition, the benign side effect of bioprinting to the printed mammalian cells can be utilized for gene or drug delivery, which can be achieved conveniently during precise cell placement for tissue construction. With layer-by-layer assembly, three-dimensional tissues with complex structures can be printed using converted medical images. Therefore, bioprinting based on thermal inkjet is so far the most optimal solution to engineer vascular system to the thick and complex tissues. Collectively, bioprinting has great potential and broad applications in tissue engineering and regenerative medicine. The future advances of bioprinting include the integration of different printing mechanisms to engineer biphasic or triphasic tissues with optimized scaffolds and further understanding of stem cell biology.     

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo^{1, 2}.In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses³.Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques⁴.This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.Open in a separate windowClick here to view.^{(27M, flv)}