Alexander Friedenstein, Mesenchymal Stem Cells, Shifting Paradigms and Euphemisms.

Six decades ago, Friedenstein and coworkers published a series of seminal papers identifying a cell population in bone marrow with osteogenic potential, now referred to as mesenchymal stem cells (MSCs). This work was also instrumental in establishing the identity of hematopoietic stem cell and the identification of skeletal stem/progenitor cell (SSPC) populations in various skeletal compartments. In recognition of the centenary year of Friedenstein's birth, I review key aspects of his work and discuss the evolving concept of the MSC and its various euphemisms indorsed by changing paradigms in the field. I also discuss the recent emphasis on MSC stromal quality attributes and how emerging data demonstrating a mechanistic link between stromal and stem/progenitor functions bring renewed relevance to Friedenstein's contributions and much needed unity to the field.

Comparative transcriptome analysis of bone marrow resident versus culture-expanded mouse mesenchymal stem/stromal cells.

BACKGROUND AIMS: Mesenchymal stem/stromal cells (MSCs) are defined as culture-expanded populations, and although these cells recapitulate many properties of bone marrow (BM) resident skeletal stem/progenitor cells, few studies have directly compared these populations to evaluate how culture adaptation and expansion impact critical quality attributes. METHODS: We analyzed by RNA sequencing Lin-SCA1+ MSCs enriched from BM by immunodepletion (ID) and after subsequent culture expansion (Ex) and Lin-LEPR+ MSCs sorted (S) directly from BM. Pairwise comparisons were used to identify differentially expressed genes (DEGs) between populations, and gene set enrichment analysis was employed to identify biological pathways/processes unique to each population. K-means cluster analysis resolved isolation status-dependent changes in transcription in pseudotime. RESULTS: Hierarchical clustering segregated populations by isolation process, and principal component analysis identified transcripts related to vasculature development, ossification and inflammatory/cytokine signaling as key drivers of population variance. Pairwise comparisons identified 3849 DEGs in ID versus S BM-MSCs mapping to Gene Ontology (GO) terms related to immune and metabolic processes and 334 DEGs in Ex versus ID BM-MSCs mapping to GO terms related to tissue development, cell growth and replication and organelle organization. K-means cluster analysis revealed significant differences in transcripts encoding stemness and differentiation markers, extracellular matrix structural constituents and remodeling enzymes and paracrine-acting factors between populations. CONCLUSIONS: These comparative analyses reveal significant differences in gene expression signatures between BM resident and culture-expanded MSCs, thereby providing new insight into how culture adaptation/expansion endows the latter with unique quality attributes.

TWIST1 and TSG6 are coordinately regulated and function as potency biomarkers in human MSCs.

Mesenchymal stem/stromal cells (MSCs) have been evaluated in >1500 clinical trials, but outcomes remain suboptimal because of knowledge gaps in quality attributes that confer potency. We show that TWIST1 directly represses TSG6 expression that TWIST1 and TSG6 are inversely correlated across bone marrow-derived MSC (BM-MSC) donor cohorts and predict interdonor differences in their proangiogenic, anti-inflammatory, and immune suppressive activity in vitro and in sterile inflammation and autoimmune type 1 diabetes preclinical models. Transcript profiling of TWIST1HiTSG6Low versus TWISTLowTSG6Hi BM-MSCs revealed previously unidentified roles for TWIST1/TSG6 in regulating cellular oxidative stress and TGF-ß2 in modulating TSG6 expression and anti-inflammatory activity. TWIST1 and TSG6 levels also correlate to donor stature and predict differences in iPSC-derived MSC quality attributes. These results validate TWIST1 and TSG6 as biomarkers that predict interdonor differences in potency across laboratories and assay platforms, thereby providing a means to manufacture MSC products tailored to specific diseases.

Graph neural networks for the identification of novel inhibitors of a small RNA.

MicroRNAs (miRNAs) play a crucial role in post-transcriptional gene regulation and have been implicated in various diseases, including cancers and lung disease. In recent years, Graph Neural Networks (GNNs) have emerged as powerful tools for analyzing graph-structured data, making them well-suited for the analysis of molecular structures. In this work, we explore the application of GNNs in ligand-based drug screening for small molecules targeting miR-21. By representing a known dataset of small molecules targeting miR-21 as graphs, GNNs can learn complex relationships between their structures and activities, enabling the prediction of potential miRNA-targeting small molecules by capturing the structural features and similarity between known miRNA-targeting compounds. The use of GNNs in miRNA-targeting drug screening holds promise for the discovery of novel therapeutic agents and provides a computational framework for efficient screening of large chemical libraries.

Transcriptional Targets of TWIST1 in Human Mesenchymal Stem/Stromal Cells Mechanistically Link Stem/Progenitor and Paracrine Functions.

Despite extensive clinical testing, mesenchymal stem/stromal cell (MSC)-based therapies continue to underperform with respect to efficacy, which reflects the paucity of biomarkers that predict potency prior to patient administration. Previously, we reported that TWIST1 predicts inter-donor differences in MSC quality attributes that confer potency. To define the full spectrum of TWIST1 activity in MSCs, the present work employed integrated omics-based profiling to identify a high-confidence set of TWIST1 targets, which mapped to cellular processes related to ECM structure/organization, skeletal and circulatory system development, interferon gamma signaling, and inflammation. These targets are implicated in contributing to both stem/progenitor and paracrine activities of MSCs indicating these processes are linked mechanistically in a TWIST1-dependent manner. Targets implicated in extracellular matrix dynamics further implicate TWIST1 in modulating cellular responses to niche remodeling. Novel TWIST1-regulated genes identified herein may be prioritized for future mechanistic and functional studies.

Revisiting the Mesenchymal "Stem vs. Stromal" Cell Dichotomy and Its Implications for Development of Improved Potency Metrics.

Mesenchymal stem/stromal cell (MSC)-based therapies have been evaluated in over 1500 human clinical trials for a diverse array of disease indication, but outcomes remain unpredictable due to knowledge gaps in the quality attributes that confer therapeutic potency onto cells and their mode of action in vivo. Based on accumulated evidence from pre-clinical models, MSCs exert therapeutic effects by repressing inflammatory and immune-mediated response via paracrine action following reprogramming by the host injury microenvironment, and by polarization of tissue resident macrophages following phagocytosis to an alternatively activated (M2) state. An important tenet of this existing paradigm is that well-established stem/progenitor functions of MSCs are independent of paracrine function and dispensable for their anti-inflammatory and immune suppressive functions. Herein, we review evidence that stem/progenitor and paracrine functions of MSCs are mechanistically linked and organized hierarchically and describe how this link may be exploited to develop metrics that predict MSC potency across a spectrum of activities and regenerative medicine applications.

Mesenchymal stem/stromal cells from a transplanted, asymptomatic patient with Fanconi anemia exhibit an aging-like phenotype and dysregulated expression of genes implicated in hematopoiesis and myelodysplasia.

BACKGROUND AIMS: Fanconi anemia (FA) is an inherited bone marrow failure syndrome caused by defects in the repair of DNA inter-strand crosslinks and manifests as aplastic anemia, myelodysplastic syndrome and acute myeloid leukemia. FA also causes defects in mesenchymal stromal cell (MSC) function, but how different FA gene mutations alter function remains understudied. METHODS: We compared the growth, differentiation and transcript profile of a single MSC isolate from an asymptomatic patient with FA with a FANCG nonsense mutation who underwent hematopoietic stem cell transplantation 10 years prior to that from a representative healthy donor (HD). RESULTS: We show that FANCG-/- MSCs exhibit rapid onset of growth cessation, skewed bi-lineage differentiation in favor of adipogenesis and increased cellular oxidate stress consistent with an aging-like phenotype. Transcript profiling identified pathways related to cell growth, senescence, cellular stress responses and DNA replication/repair as over-represented in FANCG-/- MSC, and real-time polymerase chain reaction confirmed these MSCs expressed reduced levels of transcripts implicated in cell growth (TWIST1, FGFR2v7-8) and osteogenesis (TWIST1, RUNX2) and increased levels of transcripts regulating adipogenesis (GPR116) and insulin signaling. They also expressed reduced levels of mRNAs implicated in HSC self-maintenance and homing (KITLG, HGF, GDNF, PGF, CFB, IL-1B and CSF1) and elevated levels of those implicated in myelodysplasia (IL-6, GDF15). CONCLUSIONS: Together, these findings demonstrate how inactivation of FANCG impacts MSC behavior, which parallels observed defects in osteogenesis, HSC depletion and leukemic blast formation seen in patients with FA.

Mesenchymal Stem Cell (MSC)-Derived Extracellular Vesicles Protect from Neonatal Stroke by Interacting with Microglial Cells.

Mesenchymal stem cell (MSC)-based therapies are beneficial in models of perinatal stroke and hypoxia-ischemia. Mounting evidence suggests that in adult injury models, including stroke, MSC-derived small extracellular vesicles (MSC-sEV) contribute to the neuroprotective and regenerative effects of MSCs. Herein, we examined if MSC-sEV protect neonatal brain from stroke and if this effect is mediated via communication with microglia. MSC-sEV derived from bone marrow MSCs were characterized by size distribution (NanoSight™) and identity (protein markers). Studies in microglial cells isolated from the injured or contralateral cortex of postnatal day 9 (P9) mice subjected to a 3-h middle cerebral artery occlusion (tMCAO) and cultured (in vitro) revealed that uptake of fluorescently labeled MSC-sEV was significantly greater by microglia from the injured cortex vs. contralateral cortex. The cell-type-specific spatiotemporal distribution of MSC-sEV was also determined in vivo after tMCAO at P9. MSC-sEV administered at reperfusion, either by intracerebroventricular (ICV) or by intranasal (IN) routes, accumulated in the hemisphere ipsilateral to the occlusion, with differing spatial distribution 2 h, 18 h, and 72 h regardless of the administration route. By 72 h, MSC-sEV in the IN group was predominantly observed in Iba1+ cells with retracted processes and in GLUT1+ blood vessels in ischemic-reperfused regions. MSC-sEV presence in Iba1+ cells was sustained. MSC-sEV administration also significantly reduced injury volume 72 h after tMCAO in part via modulatory effects on microglial cells. Together, these data establish feasibility for MSC-sEV delivery to injured neonatal brain via a clinically relevant IN route, which affords protection during sub-acute injury phase.

Consensus International Council for Commonality in Blood Banking Automation-International Society for Cell & Gene Therapy statement on standard nomenclature abbreviations for the tissue of origin of mesenchymal stromal cells.

The Cellular Therapy Coding and Labeling Advisory Group of the International Council for Commonality in Blood Banking Automation and the International Society for Cell & Gene Therapy mesenchymal stromal cell (MSC) committee are providing specific recommendations on abbreviating tissue sources of culture-adapted MSCs. These recommendations include using abbreviations based on the ISBT 128 terminology model that specifies standard class names to distinguish cell types and tissue sources for culture-adapted MSCs. Thus, MSCs from bone marrow are MSC(M), MSCs from cord blood are MSC(CB), MSCs from adipose tissue are MSC(AT) and MSCs from Wharton's jelly are MSC(WJ). Additional recommendations include using these abbreviations through the full spectrum of pre-clinical, translational and clinical research for the development of culture-adapted MSC products. This does not apply to basic research focused on investigating the developmental origins, identity or functionalities of endogenous progenitor cells in different tissues. These recommendations will serve to harmonize nomenclature in describing research and development surrounding culture-adapted MSCs, many of which are destined for clinical and/or commercial translation. These recommendations will also serve to align research and development efforts on culture-adapted MSCs with other cell therapy products.

Mesenchymal stem cell-derived extracellular vesicles reduce senescence and extend health span in mouse models of aging.

Aging drives progressive loss of the ability of tissues to recover from stress, partly through loss of somatic stem cell function and increased senescent burden. We demonstrate that bone marrow-derived mesenchymal stem cells (BM-MSCs) rapidly senescence and become dysfunctional in culture. Injection of BM-MSCs from young mice prolonged life span and health span, and conditioned media (CM) from young BM-MSCs rescued the function of aged stem cells and senescent fibroblasts. Extracellular vesicles (EVs) from young BM-MSC CM extended life span of Ercc1-/- mice similarly to injection of young BM-MSCs. Finally, treatment with EVs from MSCs generated from human ES cells reduced senescence in culture and in vivo, and improved health span. Thus, MSC EVs represent an effective and safe approach for conferring the therapeutic effects of adult stem cells, avoiding the risks of tumor development and donor cell rejection. These results demonstrate that MSC-derived EVs are highly effective senotherapeutics, slowing the progression of aging, and diseases driven by cellular senescence.

Mesenchymal stromal cell variables influencing clinical potency: the impact of viability, fitness, route of administration and host predisposition.

The International Society for Cell & Gene Therapy mesenchymal stromal cell (MSC) committee has been an interested observer of community interests in all matters related to MSC identity, mechanism of action, potency assessment and etymology, and it has regularly contributed to this conversation through a series of MSC pre-conferences and committee publications dealing with these matters. Arising from these reflections, the authors propose that an overlooked and potentially disruptive perspective is the impact of in vivo persistence on potency that is not predicted by surrogate cellular potency assays performed in vitro and how this translates to in vivo outcomes. Systemic delivery or extravascular implantation at sites removed from the affected organ system seems to be adequate in affecting clinical outcomes in many pre-clinical murine models of acute tissue injury and inflammatory pathology, including the recent European Medicines Agency-approved use of MSCs in Crohn-related fistular disease. The authors further propose that MSC viability and metabolic fitness likely dominate as a potency quality attribute, especially in recipients poised for salutary benefits as defined by emerging predictive biomarkers of response.

Cell-based therapies for coronavirus disease 2019: proper clinical investigations are essential.

The serious consequences of the global coronavirus disease 2019 (COVID-19) pandemic have prompted a rapid global response to develop effective therapies that can lessen disease severity in infected patients. Cell-based approaches, primarily using mesenchymal stromal cells (MSCs), have demonstrated a strong safety profile and possible efficacy in patients with acute respiratory distress syndrome (ARDS), but whether these therapies are effective for treating respiratory virus-induced ARDS is unknown. According to the World Health Organization International Clinical Trials Registry Platform and the National Institutes of Health ClinicalTrials.gov databases, 27 clinical investigations of MSC-based cell therapy approaches have begun in China since the onset of the COVID-19 outbreak, with a growing number of academic and industry trials elsewhere as well. Several recent published reports have suggested potential efficacy; however, the available data presented are either anecdotal or from incomplete, poorly controlled investigations. Therefore, although there may be a potential role for MSCs and other cell-based therapies in treatment of COVID-19, these need to be investigated in a rationally designed, controlled approach if safety and efficacy are to be demonstrated accurately. The authors urge that the field proceed by finding a balance between swift experimentation and communication of results and scientifically coherent generation and analysis of clinical data.

International Society for Extracellular Vesicles and International Society for Cell and Gene Therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19.

STATEMENT: The International Society for Cellular and Gene Therapies (ISCT) and the International Society for Extracellular Vesicles (ISEV) recognize the potential of extracellular vesicles (EVs, including exosomes) from mesenchymal stromal cells (MSCs) and possibly other cell sources as treatments for COVID-19. Research and trials in this area are encouraged. However, ISEV and ISCT do not currently endorse the use of EVs or exosomes for any purpose in COVID-19, including but not limited to reducing cytokine storm, exerting regenerative effects or delivering drugs, pending the generation of appropriate manufacturing and quality control provisions, pre-clinical safety and efficacy data, rational clinical trial design and proper regulatory oversight.

Improving mesenchymal stem/stromal cell potency and survival: Proceedings from the International Society of Cell Therapy (ISCT) MSC preconference held in May 2018, Palais des Congrès de Montréal, Organized by the ISCT MSC Scientific Committee.

As part of the International Society of Cell Therapy (ISCT) 2018 Annual Meeting, the Mesenchymal Stem/Stromal Cell (MSC) committee organized a pre-conference, which covered methods of improving MSC engraftment and potency in vivo and clinical efficacy using MSC potency assays. The speakers examined methods to improve clinical efficacy using MSC potency assays and methods to improve MSC engraftment/homing/potency in vivo. Discussion of patient "responders" versus "non-responders" in clinical trials and working toward ways to identify them were also included.

Manufacturing mesenchymal stromal cells for clinical applications: A survey of Good Manufacturing Practices at U.S. academic centers.

BACKGROUND AIMS: Mesenchymal stromal cells (MSC) have gained prominence in the field of regenerative medicine due to their excellent safety profile in human patients and recently demonstrated efficacy in late-stage clinical studies. A prerequisite to achieving successful MSC-based therapies is the development of large-scale manufacturing processes that preserve the biological potency of the founder cell population. Because no standardized manufacturing process exists for MSCs, understanding differences in these processes among U.S. academic facilities would allow for better comparison of results obtained in the clinical setting. METHODS: We collected information through a questionnaire sent to U.S. academic centers that produce MSCs under Good Manufacturing Practice conditions. RESULTS: The survey provided information on the number and geographic location of academic facilities in the United States and major trends in their manufacturing practices. For example, most facilities employed MSCs enriched from bone marrow by plastic adherence and expanded in media supplemented with pooled human platelet lysate. Sterility testing and product identification via cell surface phenotype analysis were commonly reported practices, whereas initial and working cell plating densities, culture duration, product formulation and the intended use of the MSC product were highly variable among facilities. The survey also revealed that although most facilities assessed product potency, the methods used were limited in scope compared with the broad array of intended clinical applications of the product. CONCLUSIONS: Survey responses reported herein offer insight into the current best practices used to manufacture MSC-based products in the United States and how these practices may affect product quality and potency. The responses also provide a foundation to establish standardized manufacturing platforms.

Basal p53 expression is indispensable for mesenchymal stem cell integrity.

Marrow-resident mesenchymal stem cells (MSCs) serve as a functional component of the perivascular niche that regulates hematopoiesis. They also represent the main source of bone formed in adult bone marrow, and their bifurcation to osteoblast and adipocyte lineages plays a key role in skeletal homeostasis and aging. Although the tumor suppressor p53 also functions in bone organogenesis, homeostasis, and neoplasia, its role in MSCs remains poorly described. Herein, we examined the normal physiological role of p53 in primary MSCs cultured under physiologic oxygen levels. Using knockout mice and gene silencing we show that p53 inactivation downregulates expression of TWIST2, which normally restrains cellular differentiation to maintain wild-type MSCs in a multipotent state, depletes mitochondrial reactive oxygen species (ROS) levels, and suppresses ROS generation and PPARG gene and protein induction in response to adipogenic stimuli. Mechanistically, this loss of adipogenic potential skews MSCs toward an osteogenic fate, which is further potentiated by TWIST2 downregulation, resulting in highly augmented osteogenic differentiation. We also show that p53-/- MSCs are defective in supporting hematopoiesis as measured in standard colony assays because of decreased secretion of various cytokines including CXCL12 and CSF1. Lastly, we show that transient exposure of wild-type MSCs to 21% oxygen upregulates p53 protein expression, resulting in increased mitochondrial ROS production and enhanced adipogenic differentiation at the expense of osteogenesis, and that treatment of cells with FGF2 mitigates these effects by inducing TWIST2. Together, these findings indicate that basal p53 levels are necessary to maintain MSC bi-potency, and oxygen-induced increases in p53 expression modulate cell fate and survival decisions. Because of the critical function of basal p53 in MSCs, our findings question the use of p53 null cell lines as MSC surrogates, and also implicate dysfunctional MSC responses in the pathophysiology of p53-related skeletal disorders.

Mesenchymal Stem Cells: The Moniker Fits the Science.

Mesenchymal stem cells (MSCs) have gained widespread use in regenerative medicine due to their demonstrated efficacy in a broad range of experimental animal models of disease and their excellent safety profile in human clinical trials. Outcomes from these studies suggest that MSCs achieve therapeutic effects in vivo in nonhomologous applications predominantly by paracrine action. This paracrine-centric viewpoint has become widely entrenched in the field, and has spurred a campaign to rename MSCs as "medicinal signaling cells" to better reflect this mode of action. In this Commentary, we argue that the paracrine-centric viewpoint and proposed name change ignores a wealth of old and new data that unequivocally demonstrate the stem cell nature of MSCs, and also overlooks a large effort to exploit homologous applications of MSCs in human clinical trials. Furthermore, we offer evidence that a stem cell-centric viewpoint of MSCs provides a comprehensive understanding of MSC biology that encompasses their paracrine activity, and provides a better foundation to develop metrics that quantify the biological potency of MSC batches for both homologous and nonhomologous clinical applications. Stem Cells 2018;36:7-10.

IP6K1 Reduces Mesenchymal Stem/Stromal Cell Fitness and Potentiates High Fat Diet-Induced Skeletal Involution.

Mesenchymal stem/stromal cells (MSCs) are the predominant source of bone and adipose tissue in adult bone marrow and play a critical role in skeletal homeostasis. Age-induced changes in bone marrow favor adipogenesis over osteogenesis leading to skeletal involution and increased marrow adiposity so pathways that prevent MSC aging are potential therapeutic targets for treating age-related bone diseases. Here, we show that inositol hexakisphosphate kinase 1 (Ip6k1) deletion in mice increases MSC yields from marrow and enhances cell growth and survival ex vivo. In response to the appropriate stimuli, Ip6k1-/- versus Ip6k1+/+ MSCs also exhibit enhanced osteogenesis and hematopoiesis-supporting activity and reduced adipogenic differentiation. Mechanistic-based studies revealed that Ip6k1-/- MSCs express higher MDM2 and lower p53 protein levels resulting in lower intrinsic mitochondrial reactive oxygen species (ROS) levels as compared to Ip6k1+/+ MSCs, but both populations upregulate mitochondrial ROS to similar extents in response to oxygen-induced stress. Finally, we show that mice fed a high fat diet exhibit reduced trabecular bone volume, and that pharmacological inhibition of IP6K1 using a pan-IP6K inhibitor largely reversed this phenotype while increasing MSC yields from bone marrow. Together, these findings reveal an important role for IP6K1 in regulating MSC fitness and differentiation fate. Unlike therapeutic interventions that target peroxisome proliferator-activated receptor gamma and leptin receptor activity, which yield detrimental side effects including increased fracture risk and altered feeding behavior, respectively, inhibition of IP6K1 maintains insulin sensitivity and prevents obesity while preserving bone integrity. Therefore, IP6K1 inhibitors may represent more effective insulin sensitizers due to their bone sparing properties. Stem Cells 2017;35:1973-1983.

Concise Review: MSC-Derived Exosomes for Cell-Free Therapy.

Mesenchymal stem cell transplantation is undergoing extensive evaluation as a cellular therapy in human clinical trials. Because MSCs are easily isolated and amenable to culture expansion in vitro there is a natural desire to test MSCs in many diverse clinical indications. This is exemplified by the rapidly expanding literature base that includes many in vivo animal models. More recently, MSC-derived extracellular vesicles (EVs), which include exosomes and microvesicles (MV), are being examined for their role in MSC-based cellular therapy. These vesicles are involved in cell-to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body. The exosomes and MVs can influence tissue responses to injury, infection, and disease. MSC-derived exosomes have a content that includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs. To the extent that MSC exosomes can be used for cell-free regenerative medicine, much will depend on the quality, reproducibility, and potency of their production, in the same manner that these parameters dictate the development of cell-based MSC therapies. However, the MSC exosome's contents are not static, but rather a product of the MSC tissue origin, its activities and the immediate intercellular neighbors of the MSCs. As such, the exosome content produced by MSCs appears to be altered when MSCs are cultured with tumor cells or in the in vivo tumor microenvironment. Therefore, careful attention to detail in producing MSC exosomes may provide a new therapeutic paradigm for cell-free MSC-based therapies with decreased risk. Stem Cells 2017;35:851-858.