Siddaraju V. Boregowda

Mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle microRNAs

Mesenchymal stem cells (MSCs) and macrophages are fundamental components of the stem cell niche and function coordinately to regulate haematopoietic stem cell self-renewal and mobilization. Recent studies indicate that mitophagy and healthy mitochondrial function are critical to the survival of stem cells, but how these processes are regulated in MSCs is unknown. Here we show that MSCs manage intracellular oxidative stress by targeting depolarized mitochondria to the plasma membrane via arrestin domain-containing protein 1-mediated microvesicles. The vesicles are then engulfed and re-utilized via a process involving fusion by macrophages, resulting in enhanced bioenergetics. Furthermore, we show that MSCs simultaneously shed micro RNA-containing exosomes that inhibit macrophage activation by suppressing Toll-like receptor signalling, thereby de-sensitizing macrophages to the ingested mitochondria. Collectively, these studies mechanistically link mitophagy and MSC survival with macrophage function, thereby providing a physiologically relevant context for the innate immunomodulatory activity of MSCs.

Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long‐term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2‐associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300‐fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen‐induced stress response was dependent upon p53 since siRNA‐mediated knockdown of p53 in wild‐type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long‐term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen‐induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long‐term expansion in vitro. STEM CELLS 2012;30:975–987

Pharmacological repression of PPARγ promotes osteogenesis

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) is the master regulator of adipogenesis and the pharmacological target of the thiazolidinedione (TZD) class of insulin sensitizers. Activation of PPARγ by TZDs promotes adipogenesis at the expense of osteoblast formation, contributing to their associated adverse effects on bone. Recently we reported the development of PPARγ antagonist SR1664, designed to block the obesity induced phosphorylation of serine 273 (S273) in the absence of classical agonism, to derive insulin sensitizing efficacy with improved therapeutic index. Here we identify the structural mechanism by which SR1664 actively antagonizes PPARγ, and extend these findings to develop the inverse agonist SR2595. Treatment of isolated bone marrow derived mesenchymal stem cells (MSCs) with SR2595 promotes induction of osteogenic differentiation. Together these results identify the structural determinants of ligand mediated PPARγ repression, and suggest a therapeutic approach to promote bone formation.

Therapeutic Applications of Mesenchymal Stem Cells

The past decade has seen tremendous growth in the clinical application of cell-based therapies, and the number of planned human clinical trials to evaluate these therapies continues to increase in number and scope at a rapid pace. A considerable effort on this front has been devoted to evaluating the therapeutic potential of mesenchymal stem cells (MSCs), which were initially characterized as connective tissue progenitors resident in bone marrow. MSCs are now known to possess potent tissue reparative properties that have been linked to secretion of paracrine-acting angiogenic, trophic, anti-inflammatory, and immunomodulatory factors. Accordingly, MSC-based therapies are being evaluated for the treatment of a broad array of ischemic, inflammatory, and immunological disorders. Nevertheless, knowledge regarding how the wide-ranging activities of MSCs vary between and are specified within populations remains largely unexplored. Lack of such knowledge makes it difficult to predict and/or control how sampling bias and ex vivo expansion of populations alters their biological activity and therapeutic potency. Herein, we discuss how heterogeneity of MSC populations may explain, in part, disparate outcomes in both experimental animal and human clinical trial data, and discuss several strategies to achieve more reproducible and efficacious outcomes for MSC-based therapies.

Therapeutic applications of mesenchymal stem cells: current outlook.

The past decade has seen tremendous growth in the clinical application of cell-based therapies, and the number of planned human clinical trials to evaluate these therapies continues to increase in number and scope at a rapid pace. A considerable effort on this front has been devoted to evaluating the therapeutic potential of mesenchymal stem cells (MSCs), which were initially characterized as connective tissue progenitors resident in bone marrow. MSCs are now known to possess potent tissue reparative properties that have been linked to secretion of paracrine-acting angiogenic, trophic, anti-inflammatory, and immunomodulatory factors. Accordingly, MSC-based therapies are being evaluated for the treatment of a broad array of ischemic, inflammatory, and immunological disorders. Nevertheless, knowledge regarding how the wide-ranging activities of MSCs vary between and are specified within populations remains largely unexplored. Lack of such knowledge makes it difficult to predict and/or control how sampling bias and ex vivo expansion of populations alters their biological activity and therapeutic potency. Herein, we discuss how heterogeneity of MSC populations may explain, in part, disparate outcomes in both experimental animal and human clinical trial data, and discuss several strategies to achieve more reproducible and efficacious outcomes for MSC-based therapies.

The peculiar biology of mouse mesenchymal stromal cells—oxygen is the key

Because of the ability to manipulate their genome, mice are the experimental tool of choice for many areas of scientific investigation. Moreover, established experimental mouse models of human disease are widely available and offer a valuable resource to obtain proof-of-concept for many cell-based therapies. Nevertheless, efforts to establish reliable methods to isolate mesenchymal stromal cells (MSCs) from mouse bone marrow have been elusive. Indeed, a variety of physical and genetic approaches have been described to fractionate MSCs from other cell lineages in bone marrow, but few have achieved high yields or purity while maintaining the genomic integrity of the cells. We provide a historic overview of published procedures dedicated to the isolation of mouse MSCs from bone marrow and compact bone. We also review current findings indicating that growth-restrictive conditions imposed by atmospheric oxygen promotes immortalization of mouse MSCs and how expansion in a low-oxygen environment enhances cell yields and maintains genomic stability. Finally, we provide basic recommendations for isolating primary mouse MSCs and discuss potential pitfalls associated with these isolation methods.

A Clinical Indications Prediction Scale Based on TWIST1 for Human Mesenchymal Stem Cells

In addition to their stem/progenitor properties, mesenchymal stem cells (MSCs) also exhibit potent effector (angiogenic, antiinflammatory, immuno-modulatory) functions that are largely paracrine in nature. It is widely believed that effector functions underlie most of the therapeutic potential of MSCs and are independent of their stem/progenitor properties. Here we demonstrate that stem/progenitor and effector functions are coordinately regulated at the cellular level by the transcription factor Twist1 and specified within populations according to a hierarchical model. We further show that manipulation of Twist1 levels by genetic approaches or by exposure to widely used culture supplements including fibroblast growth factor 2 (Ffg2) and interferon gamma (IFN-gamma) alters MSC efficacy in cell-based and in vivo assays in a predictable manner. Thus, by mechanistically linking stem/progenitor and effector functions our studies provide a unifying framework in the form of an MSC hierarchy that models the functional complexity of populations. Using this framework, we developed a CLinical Indications Prediction (CLIP) scale that predicts how donor-to-donor heterogeneity and culture conditions impact the therapeutic efficacy of MSC populations for different disease indications.

Quantifiable Metrics for Predicting MSC Therapeutic Efficacy

Currently there are over 600 clinical trials listed at www. clinicaltrials.gov utilizing Mesenchymal Stromal Cells (MSCs) as an experimental cell-based therapy, making MSCs the most commonly employed cell type under investigation for treating human diseases. MSCs have gained widespread use in regenerative medicine due to their demonstrated potency in a broad range of experimental animal models of disease and their excellent safety profile in human clinical trials. However, while MSC-based therapies have clearly shown benefits in patients with ischemic and immune-related disorders, many trials completed to date have yielded suboptimal outcomes and several have failed to meet their primary endpoints of efficacy. A challenge in the development of efficacious MSC-based therapies is the inability to consistently manufacture homogeneous populations of cells with known efficacy for a specific disease indication that yield predictable and reproducible patient outcomes. This difficulty stems from the fact that methods routinely used to isolate MSCs [1–4] yield populations that exhibit significant heterogeneity in terms of morphologic features, growth rate, life span, differentiation potential, and potency in functional-based assays [5–8]. Donor-to-donor heterogeneity coupled with the lack of standardized manufacturing protocols makes it impossible to determine if patients enrolled in different clinical trials received functionally equivalent MSC preparations. The lack of metrics that discriminate functional differences between populations further confounds efforts to select the most suitable populations for a given disease indication. Therefore, the identification and reduction to practice of manufacturing schemes with deployable metrics to assess efficacy prior to patient administration is necessary to improve clinical outcomes and advance MSCbased therapies beyond the experimental phase.

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