Regenerative Cells for the Management of Osteoarthritis and Joint Disorders: A Concise Literature Review.

As the global population ages, the prevalence of osteoarthritis (OA) and joint disorders represent a major cause of disability and a significant public health burden. As current approaches for the management of OA focus on slowing the progression of disease, without repairing the underlying damage, novel treatments are necessary to improve outcomes. Over the past decade, autologous cell-based therapies using regenerative cells from fat or bone marrow have become a major focus of research into new approaches for the treatment of osteoarthritis and joint disorders. This review is intended to summarize findings in existing literature and identify gaps in knowledge that should be addressed in order to advance the field. We acknowledge that some findings may appear inconsistent, but show that apparent inconsistency in the literature may be attributable to variation in source of cells, stage of disease, method of delivery, follow-up time, evaluation method, and a number of other idiosyncrasies of individual studies. Still, a number of themes emerge from the data and some broader conclusions may be drawn that can be used to guide future studies. Ultimately, we conclude that there is overwhelming evidence demonstrating the safety of the autologous cell-based therapies. Furthermore, the data support the claim that regenerative cells are capable of reversing progression of OA. Regenerative cells, and especially those from adipose tissue, represent a promising new approach for the treatment of OA. Future work should include appropriate controls, and focus on answering questions related to dose required, appropriate delivery vehicle, and the impact of multiple treatments. Additionally, future studies should look at short and long-term effects of the treatments, and use functional as well as radiologic methods to evaluate efficacy.

Matrix-bound Cyr61/CCN1 is required to retain the properties of the bone marrow mesenchymal stem cell niche but is depleted with aging.

Previously, we showed that extracellular matrices (ECMs), produced ex vivo by various types of stromal cells, direct bone marrow mesenchymal stem cells (BM-MSCs) in a tissue-specific manner and recapitulate physiologic changes characteristic of the aging microenvironment. In particular, BM-MSCs obtained from elderly donors and cultured on ECM produced by young BM stromal cells showed improved quantity, quality and osteogenic differentiation. In the present study, we searched for matrix components that are required for a functional BM-MSC niche by comparing ECMs produced by BM stromal cells from "young" (≤25 y/o) versus "elderly" (≥60 y/o) donors. With increasing donor age, ECM fibrillar organization and mechanical integrity deteriorated, along with the ability to promote BM-MSC proliferation and responsiveness to growth factors. Proteomic analyses revealed that the matricellular protein, Cyr61/CCN1, was present in young, but undetectable in elderly, BM-ECM. To assess the role of Cyr61 in the BM-MSC niche, we used genetic methods to down-regulate the incorporation of Cyr61 during production of young ECM and up-regulate its incorporation in elderly ECM. The results showed that Cyr61-depleted young ECM lost the ability to promote BM-MSC proliferation and growth factor responsiveness. However, up-regulating the incorporation of Cyr61 during synthesis of elderly ECM restored its ability to support BM-MSC responsiveness to osteogenic factors such as BMP-2 and IGF-1. We next examined aging bone and compared bone mineral density and Cyr61 content of L4-L5 vertebral bodies in "young" (9-11 m/o) and "elderly" (21-33 m/o) mice. Our analyses showed that low bone mineral density was associated with decreased amounts of Cyr61 in osseous tissue of elderly versus young mice. Our results strongly demonstrate a novel role for ECM-bound Cyr61 in the BM-MSC niche, where it is responsible for retention of BM-MSC proliferation and growth factor responsiveness, while depletion of Cyr61 from the BM niche contributes to an aging-related dysregulation of BM-MSCs. Our results also suggest new potential therapeutic targets for treating age-related bone loss by restoring specific ECM components to the stem cell niche.

Oral and Craniofacial Stem Cells: An Untapped Source for Neural Tissue Regeneration.

Nerve tissue regeneration continues to represent an intractable obstacle to realizing the promise of tissue engineering. Although neurobiology works to shed light on the mechanisms governing neuronal growth and repair, considerable technical gaps remain that hinder progress. Chief among these is the absence of an appropriate culture environment to faithfully reproduce the neuronal niche ex vivo. We propose that the various multipotent cells found in the oral cavity may represent an important yet underutilized resource for preparing such neurogenic microenvironments. Similar to those of nerve tissue, these cell populations are of ectodermal origin and have clinically demonstrated neurogenic potential. Although there is a lack of consensus on whether putative types of oral and craniofacial stem cells constitute distinct populations, their contribution to neural tissue engineering may be twofold: as a cellular feedstock for neoneurogenesis and for the production of specialized in vitro environments for neurogenic differentiation, phenotype maintenance, and use in therapeutic applications. Impact statement We propose that addressing gaps in understanding the neurogenic role of dental stem cells and their microenvironment may yield efficient and reliable strategies for long-term neuronal cell culture and open new avenues for neural regeneration in both dental, nerve, and other tissues.

Native extracellular matrix, synthesized ex vivo by bone marrow or adipose stromal cells, faithfully directs mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) are highly responsive to cues in the microenvironment (niche) that must be recapitulated ex vivo to study their authentic behavior. In this study, we hypothesized that native bone marrow (BM)- and adipose (AD)-derived extracellular matrices (ECM) were unique in their ability to control MSC behavior. To test this, we compared proliferation and differentiation of bone marrow (BM)-derived MSCs when maintained on native decellularized ECM produced by BM versus AD stromal cells (i.e. BM- versus AD-ECM). We found that both ECMs contained similar types of collagens but differed in the relative abundance of each. Type VI collagen was the most abundant (≈60% of the total collagen present), while type I was the next most abundant at ≈30%. These two types of collagen were found in nearly equal proportions in both ECMs. In contrast, type XII collagen was almost exclusively found in AD-ECM, while types IV and V were only found in BM-ECM. Physically and mechanically, BM-ECM was rougher and stiffer, but less adhesive, than AD-ECM. During 14 days in culture, both ECMs supported BM-MSC proliferation better than tissue culture plastic (TCP), although MSC-related surface marker expression remained relatively high on all three culture surfaces. BM-MSCs cultured in osteogenic (OS) differentiation media on BM-ECM displayed a significant increase in calcium deposition in the matrix, indicative of osteogenesis, while BM-MSCs cultured on AD-ECM in the presence of adipogenic (AP) differentiation media showed a significant increase in Oil Red O staining, indicative of adipogenesis. Further, culture on BM-ECM significantly increased BM-MSC-responsiveness to rhBMP-2 (an osteogenic inducer), while culture on AD-ECM enhanced responsiveness to rosiglitazone (an adipogenic inducer). These findings support our hypothesis and indicate that BM- and AD-ECMs retain unique elements, characteristic of their tissue-specific microenvironment (niche), which promote retention of MSC differentiation state (i.e. "stemness") during expansion and direct cell response to lineage-specific inducers. This study provides a new paradigm for precisely controlling MSC fate to a desired cell lineage for tissue-specific cell-based therapies.

Restoring the quantity and quality of elderly human mesenchymal stem cells for autologous cell-based therapies.

BACKGROUND: Degenerative diseases are a major public health concern for the aging population and mesenchymal stem cells (MSCs) have great potential for treating many of these diseases. However, the quantity and quality of MSCs declines with aging, limiting the potential efficacy of autologous MSCs for treating the elderly population. METHODS: Human bone marrow (BM)-derived MSCs from young and elderly donors were obtained and characterized using standard cell surface marker criteria (CD73, CD90, CD105) as recommended by the International Society for Cellular Therapy (ISCT). The elderly MSC population was isolated into four subpopulations based on size and stage-specific embryonic antigen-4 (SSEA-4) expression using fluorescence-activated cell sorting (FACS), and subpopulations were compared to the unfractionated young and elderly MSCs using assays that evaluate MSC proliferation, quality, morphology, intracellular reactive oxygen species, ß-galactosidase expression, and adenosine triphosphate (ATP) content. RESULTS: The ISCT-recommended cell surface markers failed to detect any differences between young and elderly MSCs. Here, we report that elderly MSCs were larger in size and displayed substantially higher concentrations of intracellular reactive oxygen species and ß-galactosidase expression and lower amounts of ATP and SSEA-4 expression. Based on these findings, cell size and SSEA-4 expression were used to separate the elderly MSCs into four subpopulations by FACS. The original populations (young and elderly MSCs), as well as the four subpopulations, were then characterized before and after culture on tissue culture plastic and BM-derived extracellular matrix (BM-ECM). The small SSEA-4-positive subpopulation representing ~ 8% of the original elderly MSC population exhibited a "youthful" phenotype that was similar to that of young MSCs. The biological activity of this elderly subpopulation was inhibited by senescence-associated factors produced by the unfractionated parent population. After these "youthful" cells were isolated and expanded (three passages) on a "young microenvironment" (i.e., BM-ECM produced by BM cells from young donors), the number of cells increased ≈ 17,000-fold to 3 × 109 cells and retained their "youthful" phenotype. CONCLUSIONS: These results suggest that it is feasible to obtain large numbers of high-quality autologous MSCs from the elderly population and establish personal stem cell banks that will allow serial infusions of "rejuvenated" MSCs for treating age-related diseases.

Moderate modulation of disease in the G93A model of ALS by the compound 2-(2-hydroxyphenyl)-benzoxazole (HBX).

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurological disease characterized by degeneration and death of motor neurons. Aberrant protein aggregation and oxidative stress are implicated in the etiology of ALS; thus preventing propagation of early aggregation events and oxidative damage could be an effective therapy. We tested the effect of dietary supplementation (initiated 40 days of age) with 2-(2-hydroxyphenyl)-benzoxazole (HBX), a compound with metal chelator and anti-aggregation properties, on disease onset, progression and lifespan in the G93A mouse model of ALS. Tests were not sufficiently powerful to detect any change to survival distribution of mice treated with HBX. However, the disease onset was delayed and max lifespan was increased in the treatment group. Additionally, disease progression was moderated as shown by reduced neuromuscular denervation measured by repetitive nerve stimulation. F2-isoprostanes, a marker of oxidative damage, are elevated in skeletal muscle from G93A mice at onset and this increase is prevented in HBX fed G93A mice. Furthermore, HBX treatment reduced mutant SOD1 protein aggregation in whole spinal cord of G93A mice at disease onset. Overall, our data suggests that HBX may be able to improve the degenerative symptoms of ALS through the prevention of oxidative damage and protein aggregation. Further studies are needed to uncover the mechanistic effects of HBX in ameliorating ALS pathology.

One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior.

For more than 100years, cells and tissues have been studied in vitro using glass and plastic surfaces. Over the last 10-20years, a great body of research has shown that cells are acutely sensitive to their local environment (extracellular matrix, ECM) which contains both chemical and physical cues that influence cell behavior. These observations suggest that modern cell culture systems, using tissue culture polystyrene (TCP) surfaces, may fail to reproduce authentic cell behavior in vitro, resulting in "artificial outcomes." In the current study, we use bone marrow (BM)- and adipose (AD)-derived stromal cells to prepare BM-ECM and AD-ECM, which are decellularized after synthesis by the cells, to mimic the cellular niche for each of these tissues. Each ECM was characterized for its ability to affect BM- and AD-mesenchymal stem cell (MSC) proliferation, as well as proliferation of three cancer cell lines (HeLa, MCF-7, and MDA-MB-231), modulate cell spreading, and direct differentiation relative to standard TCP surfaces. We found that both ECMs promoted the proliferation of MSCs, but that this effect was enhanced when the tissue-origin of the cells matched that of the ECM (i.e. BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM were shown to preferentially direct MSC differentiation towards either osteogenic or adipogenic lineage, respectively, suggesting that the effects of the ECM were tissue-specific. Further, each ECM influenced cell morphology (i.e. circularity), irrespective of the origin of the MSCs, lending more support to the idea that effects were tissue specific. Interestingly, unlike MSCs, these ECMs did not promote the proliferation of the cancer cells. In an effort to further understand how these three culture substrates influence cell behavior, we evaluated the chemical (protein composition) and physical properties (architecture and mechanical) of the two ECMs. While many structural proteins (e.g. collagen and fibronectin) were found at equivalent levels in both BM- and AD-ECM, the architecture (i.e. fiber orientation; surface roughness) and physical properties (storage modulus, surface energy) of each were unique. These results, demonstrating differences in cell behavior when cultured on the three different substrates (BM- and AD-ECM and TCP) with differences in chemical and physical properties, provide evidence that the two ECMs may recapitulate specific elements of the native stem cell niche for bone marrow and adipose tissues. More broadly, it could be argued that ECMs, elaborated by cells ex vivo, serve as an ideal starting point for developing tissue-specific culture environments. In contrast to TCP, which relies on the "one size fits all" paradigm, native tissue-specific ECM may be a more rational model to approach engineering 3D tissue-specific culture systems to replicate the in vivo niche. We suggest that this approach will provide more meaningful information for basic research studies of cell behavior as well as cell-based therapeutics.

Umbilical cord blood-derived non-hematopoietic stem cells retrieved and expanded on bone marrow-derived extracellular matrix display pluripotent characteristics.

BACKGROUND: Umbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs), but also non-hematopoietic stem cells (NHSCs) that are able to differentiate into a number of distinct cell types. Based on studies published to date, the frequency of NHSCs in UCB is believed to be very low. However, the isolation of these cells is primarily based on their adhesion to tissue culture plastic surfaces. METHODS AND RESULTS: In the current study, we demonstrate that this approach overlooks some of the extremely immature NHSCs because they lack the ability to adhere to plastic. Using a native extracellular matrix (ECM), produced by bone marrow (BM) stromal cells, the majority of the UCB-NHSCs attached within 4 h. The colony-forming unit fibroblast frequency of these cells was 1.5 × 104/108 mononuclear cells, which is at least 4000-fold greater than previously reported for UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration. CONCLUSIONS: These results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications.

Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions.

INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions. METHODS: Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates. RESULTS: Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates. CONCLUSIONS: Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.