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.

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.

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.

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.

Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels.

Mesenchymal stem cells (MSCs) are known to differentiate into connective tissue lineages but intracellular signaling pathways that maintain cells in an undifferentiated state remain largely unexplored. Previously, we reported that fibroblast growth factor 2 (Fgf2) reversibly inhibited multilineage differentiation of primary mouse MSCs and now identify a unique compliment of signaling proteins that are dynamically regulated by this mitogen and whose expression levels are strongly correlated with inhibition of cell differentiation. Fgf2 selectively induced expression of Twist2 and Sprouty4 (Spry4) and repressed expression of soluble frizzled related receptor 2 (Sfrp2), runt-related transcription factor 2 (Runx2), and peroxisome proliferation activated receptor gamma (Pparg). In contrast, Wnt3a induced expression of Twist but not Twist2 or Spry4 and bone morphogenetic protein 2 (Bmp2) failed to alter expression of all three genes. Moreover, pretreatment of MSCs with Fgf2 delayed extracellular regulated kinase 1 (Erk1) and Erk2 phosphorylation and repressed bone-specific gene expression during an osteoinduction time course. Alternatively, pretreatment with Wnt3a had no effect, whereas Bmp2 pretreatment augmented Erk1/2 activation and bone-specific gene expression. Fgf2 also induced expression of Fgf receptor 1 (Fgfr1) and Fgfr4 and repressed Fgfr2 and Fgfr3 expression in MSCs, whereas Wnt3a and Bmp2 had the opposite effect. Finally, immunostaining revealed that Twist and Spry4 were coexpressed in MSCs and that Fgf2 treatment altered their subcellular distribution in a manner consistent with their mode of action. Collectively, these studies demonstrate that inhibition of mouse MSC differentiation by Fgf2 is strongly correlated with upregulation of Twist2 and Spry4 and suppression of Erk1/2 activation.

Breast Masses Detection and Segmentation in Full-Field Digital Mammograms using Unified Convolution Neural Network.

Breast Cancer has been the primary reason for mortality in women of age between twenties and sixties worldwide; moreover early detection and treatment provides patients to get absolute treatment and decrease the mortality rate. Furthermore, recent research indicates that most experienced physicians have plenty of limitations, hence the plethora of work has been carried out to develop an automated mechanism of segmentation and classification of affected area and type of cancer; however, it is still considered to be highly challenging due to the variability of tumor in shape, low signal to noise ratio, shape, size and location of tumor. Furthermore, mammographic mass segmentation and detection are performed as a separate task and a convolution neural network is a highly adopted architecture for the same. In this research, we have designed and developed unified CNN architecture to perform the segmentation and detection of a breast mass. The unified-CNN architecture comprises a novel module for convolution which is combined through additional offset. Further RRS aka Random Region Selection mechanism is applied for data augmentation approach and high-level feature map is implied to achieve the high prediction. Furthermore, unified-CNN is evaluated using the metrics like true positive Rate at FPI (False Positive per Image) and Dice Index on INBreast dataset, also comparative analysis is out carried with various existing methodology. Unified-CNN is developed through improvising CNN. It introduces a novel module at the convolution layer to aim for a high-level feature map in order to get a high prediction. RRS (Random Region Selection) algorithm is used as the data augmentation approach to select the boundary region of the affected area; further robust model training is designed and optimized for process to make optimal. Unified-CNN introduces novel module at the convolution layer to aim for high level feature map in order to get high prediction; further ROI pooling is utilized for boundary detection in images.

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.

Isolation of Mouse Bone Marrow Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) were initially characterized as connective tissue progenitors resident in bone marrow, but have now been isolated from a variety of tissues and organs and shown to also exhibit potent tissue regenerative properties mediated largely via paracrine actions. These findings have spurred the development of MSC-based therapies for treating a diverse array of nonskeletal diseases. Although genetic and experimental rodent models of disease represent important tools for developing efficacious MSC-based therapies, development of reliable methods to isolate MSCs from mouse bone marrow has been hampered by the unique biological properties of these cells. Indeed, few isolation schemes afford high yields and purity while maintaining the genomic integrity of cells. We recently demonstrated that mouse MSCs are highly sensitive to oxidative stress, and long-term expansion of these cells in atmospheric oxygen selects for immortalized clones that lack a functional p53 protein. Herein, we describe a protocol for the isolation of primary MSCs from mouse bone marrow that couples immunodepletion with culture in a low-oxygen environment and affords high purity and yield while preserving p53 function.

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.

Allo-reactivity of mesenchymal stem cells in rhesus macaques is dose and haplotype dependent and limits durable cell engraftment in vivo.

The emerging paradigm that MSCs are immune privileged has fostered the use of "off-the-shelf" allogeneic MSC-based therapies in human clinical trials. However, this approach ignores studies in experimental animals wherein transplantation of MSCs across MHC boundaries elicits measurable allo-immune responses. To determine if MSCs are hypo-immunogeneic, we characterized the immune response in rhesus macaques following intracranial administration of allogeneic vs. autologous MSCs. This analysis revealed unambiguous evidence of productive allo-recognition based on expansion of NK, B and T cell subsets in peripheral blood and detection of allo-specific antibodies in animals administered allogeneic but not autologous MSCs. Moreover, the degree of MHC class I and II mismatch between the MSC donor and recipient significantly influenced the magnitude and nature of the allo-immune response. Consistent with these findings, real-time PCR analysis of brain tissue from female recipients administered varying doses of male, allogeneic MSCs revealed a significant inverse correlation between MSC engraftment levels and cell dose. Changes in post-transplant neutrophil and lymphocyte counts also correlated with dose and were predictive of overall MSC engraftment levels. However, secondary antigen challenge failed to elicit a measurable immune response in allogeneic recipients. Finally, extensive behavior testing of animals revealed no main effect of cell dose on motor skills, social development, or temperament. Collectively, these data indicate that allogeneic MSCs are weakly immunogenic when transplanted across MHC boundaries in rhesus macaques and this negatively impacts durable engraftment levels. Therefore the use of unrelated donor MSCs should be carefully evaluated in human patients.