Arnold I. Caplan

Amino acids are the essential building block for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins require uptake of various essential/non-essential amino acids. Analyzing amino acids metabolism during chondrogenesis can help to relate tissue quality to amino acids metabolism under different conditions. In our study, we studied amino acids uptake/secretion using hMSC-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acids uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized GAG and HYP content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results showed that amino acids uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of neonatal cartilage or adult cartilage. Further, this deficiency is likely related to the downregulation of some key amino acid transporters on the cells. Such deficiency could be partially improved by increasing the amino acid availability in chondrogenic medium by changing culture conditions.

The focus of this Commentary is to introduce cell-based therapy in the context of how I believe the U.S. Food and Drug Administration (FDA) might establish criteria for the approval of clinical trials that could eventually lead to the final marketplace approval of these medically relevant, cell-based therapeutic products. It is important to emphasize that regulatory agencies have set up practices and procedures that are based on many years of evaluating pharmaceutically provided drugs. To consider cell-based therapies as single action drugs is inappropriate given the complexity of this technology. The regulatory agencies have been slowly reevaluating the criteria by which they allow clinical trials using cell-based therapies to proceed. This commentary focuses on a few key aspects of such considerations and provides suggestions for modifying the standard criteria.

Human Mesenchymal Stem Cell (hMSC) immunotherapy has been shown to provide both anti-inflammatory and anti-microbial effectiveness in a variety of diseases. The clinical potency of hMSCs is based upon an initial direct hMSC effect on the pro-inflammatory and anti-microbial pathophysiology as well as sustained potency through orchestrating the host immunity to optimize the resolution of infection and tissue damage. Cystic fibrosis (CF) patients suffer from a lung disease characterized by excessive inflammation and chronic infection as well as a variety of other systemic anomalies associated with the consequences of abnormal cystic fibrosis transmembrane conductance regulator (CFTR) function. The application of hMSC immunotherapy to the CF clinical armamentarium is important even in the era of modulators when patients with an established disease still need anti-inflammatory and anti-microbial therapies. Additionally, people with CF mutations not addressed by current modulator resources need anti-inflammation and anti-infection management. Furthermore, hMSCs possess dynamic therapeutic properties, but the potency of their products is highly variable with respect to their anti-inflammatory and anti-microbial effects. Due to the variability of hMSC products, we utilized standardized in vitro and in vivo models to select hMSC donor preparations with the greatest potential for clinical efficacy. The models that were used recapitulate many of the pathophysiologic outcomes associated with CF. We applied this strategy in pursuit of identifying the optimal donor to utilize for the “First in CF” Phase I clinical trial of hMSCs as an immunotherapy and anti-microbial therapy for people with cystic fibrosis. The hMSCs screened in this study demonstrated significant diversity in antimicrobial and anti-inflammatory function using models which mimic some aspects of CF infection and inflammation. However, the variability in activity between in vitro potency and in vivo effectiveness continues to be refined. Future studies require and in-depth pursuit of hMSC molecular signatures that ultimately predict the capacity of hMSCs to function in the clinical setting.

White adipose tissues are major endocrine organs that release factors, termed adipokines, which affect other major organ systems. The development and functions of adipose tissues depend largely upon the glycosaminoglycan heparan sulfate. Heparan sulfate proteoglycans (HSPGs) surround both adipocytes and vascular structures and facilitate the communication between these two components. This communication mediates the continued export of adipokines from adipose tissues. Heparan sulfates regulate cellular physiology and communication through a sulfation code that ionically interacts with heparan-binding regions on a select set of proteins. Many of these proteins are growth factors and chemokines that regulate tissue function and inflammation. Cells regulate heparan sulfate sulfation through the release of heparanases and sulfatases. It is now possible to tissue engineer vascularized adipose tissues that express heparan sulfate proteoglycans. This makes it possible to use these tissue constructs to study the role of heparan sulfates in the regulation of adipokine production and release. It is possible to regulate the production of heparanases and sulfatases in order to fine-tune experimental studies.

The production of a clinically useful engineered cartilage is an outstanding and unmet clinical need. High throughput RNA sequencing provides a means of characterizing the molecular phenotype of populations of cells and can be leveraged to better understand differences amongst source cells, derivative engineered tissues, and target phenotypes. In this study, small RNA sequencing is utilized to comprehensively characterize the microRNA transcriptomes (miRNomes) of native human neonatal articular cartilage and human bone marrow derived mesenchymal stem cells (hBM-MSCs) differentiating into cartilage organoids, contrasting the microRNA regulation of engineered cartilage with that of a promising target phenotype. Five dominant microRNAs are upregulated during cartilage organoid differentiation and disproportionately regulate transcription factors: miR-148a-3p, miR-140-3p, miR-27b-3p, miR-140-5p, and miR-181a-5p. Two microRNAs that dominate the miRNomes of hBM-MSCs, miR-21-5p and miR-143-3p, persist throughout the differentiation process and may limit the ability of these cells to differentiate into an engineered cartilage resembling target native articular cartilage. By using predictive bioinformatics tools and antagomir inhibition, these persistent microRNAs are shown to destabilize the mRNA of genes with known or potential roles in cartilage biology including FGF18, TGFBR2, TET1, STOX2, ARAP2, N4BP2L1, LHX9, NFIA, and RPS6KA5. These results shed light on the extent to which only a few microRNAs contribute to the complex regulatory environment of hBM-MSCs for engineered tissues.

Mesenchymal stem cells were developed as a cell-based therapeutic in the 1990's. The translation of culture expanded mesenchymal stem cells from a basic science focus into a modern therapeutic has taken 30 years. The current state of the basic science information argues that mesenchymal stem cells may be curative for coronavirus disease 2019 (COVID-19). Indeed, early small-scale clinical trials have shown positive results. The issue raised is how to assemble the resources to get this cell-based therapy approved for clinical use. The technology is complex, the COVID-19 viral infections are life threatening, the cost is high, but human life is precious. What will it take to perfect this potentially curative technology?

Understanding in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) is important as it holds great promise for cartilage tissue engineering and other applications. However, the end tissue quality is highly variable and dependent on culture conditions. We investigated the effect of nutrient availability on hMSCs chondrogenesis in a static aggregate culture system by varying the medium-change frequency together with starting glucose levels. Glucose uptake and lactate secretion profiles were obtained to monitor the metabolism change during hMSCs chondrogenesis among different culture conditions. Higher medium-change frequency led to increases in cumulative glucose uptake for all starting glucose levels. Further, increase in glucose uptake by aggregates led to increased end tissue GAG and HYP content. The results suggest that increased glucose availability either via increased medium-change frequency and higher initial glucose levels lead to improved chondrogenesis. Further, cumulative glucose uptake and lactate secretion were found to correlate well with GAG and HYP content, indicating both molecules are promising biomarkers for non-invasive assessment of hMSCs chondrogenesis. Collectively, our results can be used to design optimal culture conditions and to develop dynamic assessment strategies for cartilage tissue engineering applications.

Bone marrow derived adult human mesenchymal stem cells (hMSCs) possess therapeutic qualities that enable them to enhance wound repair. However, the mechanisms by which this occurs remains poorly understood. Basic mechanisms may include the directed migration of delivered cells to target sites and/or the production and release of soluble factors that act at a distance. Allogeneic and even xenogeneic cells may effectively participate in wound repair. Labeled hMSCs were delivered to full-thickness skin wounds that were created in immunologically competent mice. The delivery occurred on day 3 post-wounding using two different carriers; one which released cells and one which retained cells. The fates of the delivered cells were tracked for up to 25 days. During this period, released cells migrated as a tight cohort deep into the wound to reach the subdermal vascular plexus. Simultaneously, enhanced formation of granulation tissue was evident. This migration of hMSCs was not essential in that enhanced granulation tissue formation and wound contraction occurred when cells were retained in the carrier matrix. This provided further evidence for the release of therapeutic factors by hMSCs to sites of injury.

Chronic nontuberculous mycobacterial infections with Mycobacterium avium and Mycobacterium intracellulare complicate bronchiectasis, chronic obstructive airway disease, and the health of aging individuals. These insidious intracellular pathogens cause considerable morbidity and eventual mortality in individuals colonized with these bacteria. Current treatment regimens with antibiotic macrolides are both toxic and often inefficient at providing infection resolution. In this article, we demonstrate that human marrow-derived mesenchymal stem cells are antimicrobial and anti-inflammatory in vitro and in the context of an in vivo sustained infection of either M. avium and/or M. intracellulare.