Multi-omic analysis of mandibuloacral dysplasia type A patient iPSC-derived MSC senescence reveals miR-311 as a novel biomarker for MSC senescence.

Mandibuloacral dysplasia type A (MADA) is a rare genetic progeroid syndrome associated with lamin A/C (LMNA) mutations. Pathogenic mutations of LMNA result in nuclear structural abnormalities, mesenchymal tissue damage and progeria phenotypes. However, it remains elusive how LMNA mutations cause mesenchymal-derived cell senescence and disease development. Here, we established an in vitro senescence model using induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) from MADA patients with homozygous LMNA p.R527C mutation. When expanded to passage 13 in vitro, R527C iMSCs exhibited marked senescence and attenuation of stemness potential, accompanied by immunophenotypic changes. Transcriptome and proteome analysis revealed that cell cycle, DNA replication, cell adhesion and inflammation might play important roles in senescence. In-depth evaluation of changes in extracellular vesicle (EV) derived iMSCs during senescence revealed that R527C iMSC-EVs could promote surrounding cell senescence by carrying pro-senescence microRNAs (miRNAs), including a novel miRNA called miR-311, which can serve as a new indicator for detecting chronic and acute mesenchymal stem cell (MSC) senescence and play a role in promoting senescence. Overall, this study advanced our understanding of the impact of LMNA mutations on MSC senescence and provided novel insights into MADA therapy as well as the link between chronic inflammation and aging development.

Rps6ka2 enhances iMSC chondrogenic differentiation to attenuate knee osteoarthritis through articular cartilage regeneration in mice.

Articular cartilage (AC) is most susceptible to degeneration in knee osteoarthritis (OA); however, the existing treatments for OA do not target the core link of the pathogenesis-"decreased tissue cell function activity and extracellular matrix (ECM) metabolic disorders" for effective intervention. iMSC hold lower heterogeneity and great promise in biological research and clinical applications. Rps6ka2 may play an important role in the iMSC to treat OA. In this study, the CRISPR/Cas9 gene editing Rps6ka2-/- iMSC were obtained. Effect of Rps6ka2 on iMSC proliferation and chondrogenic differentiation was evaluated in vitro. An OA model was constructed in mice by surgical destabilization of medial meniscus (DMM). The Rps6ka2-/- iMSC and iMSC were injected into the articular cavity twice-weekly for 8 weeks. In vitro experiments showed that Rps6ka2 could promote iMSC proliferation and chondrogenic differentiation. In vivo results further confirmed that Rps6ka2 could improve iMSC viability to promote ECM production to attenuate OA in mice.

Induced Pluripotent Stem Cells Detection via Ensemble Yolo Network.

Induced pluripotent stem cells (iPSCs) have huge potential in regenerative medicine research and industrial applications. However, building automatic method without using cell staining technique for iPSCs identification is an important challenge. To improve the efficiency of producing iPSCs, we build an accurate and noninvasive iPSCs colonies detection method via ensemble Yolo network based on the self-collected bright-field microscopy images. Meanwhile, test-time augmentation (TTA) is leveraged to further improve the detection result of our iPSCs colonies detection method. Extensive experimental results on our dataset demonstrate that our method obtains quite favorable detection performance with the highest F1 score of 0.867 and the highest mean average precision score of 0.898, which outperforms most mainstream methods.

Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells Hold Lower Heterogeneity and Great Promise in Biological Research and Clinical Applications.

Mesenchymal stem cells (MSC) isolated from different tissue sources exhibit multiple biological effects and have shown promising therapeutic effects in a broad range of diseases. In order to fulfill their clinical applications in context of precision medicine, however, more detailed molecular characterization of diverse subgroups and standardized scalable production of certain functional subgroups would be highly desired. Thus far, the generation of induced pluripotent stem cell (iPSC)-derived MSC (iMSC) seems to provide the unique opportunity to solve most obstacles that currently exist to prevent the broad application of MSC as an advanced medicinal product. The features of iMSC include their single cell clone origins, and defined and controllable cultural conditions for their derivation and proliferation. Still, comprehensive research of the molecular and functional heterogeneity of iMSC, just like MSC from any other tissue types, would be required. Furthered on previous efforts on iMSC differentiation and expansion platform and transcriptomic studies, advantages of single cell multi-omics analysis and other up-to-dated technologies would be taken in order to elucidate the molecular origin and regulation of heterogeneity and to obtain iMSC subgroups homogeneous enough for particular clinical conditions. In this perspective, the current obstacles in MSC applications, the advantages of iMSC over MSC and their implications for biological research and clinical applications will be discussed.

Quality evaluation of induced pluripotent stem cell colonies by fusing multi-source features.

BACKGROUND AND OBJECTIVE: Induced pluripotent stem cells (iPSCs) have great potential as the basis of regenerative medicine. In this paper, we propose an automatic quality evaluation model based on multi-source feature ensemble learning to divide the iPSC colonies into three categories: good, medium and bad. METHODS: First, we obtained iPSCs samples using a Sendai virus reprogramming method. Second, we collected the bright field-images of iPSC colonies and processed them with adaptive gamma transform and data enhancement. The evaluation for the iPSC colony quality was further verified with living cell fluorescent staining, currently accepted as the optimal biological method. Third, multi-source features were extracted using three deep convolutional neural networks (DCNNs) and four traditional feature descriptors. Finally, we utilized a support vector machine (SVM) to perform classification. Before feeding into the SVM, the features were processed by principal component analysis algorithm to save computational cost and training time. RESULTS: Experimental results on the collected iPSC dataset (46,500 images) show that the proposed method could obtain 95.55% classification accuracy. CONCLUSIONS: Our study could provide a method to efficiently and quickly judge the biological quality of a single iPSC colony or populations and facilitate the large-scale iPSC manufacturing.

Kartogenin mediates cartilage regeneration by stimulating the IL-6/Stat3-dependent proliferation of cartilage stem/progenitor cells.

A decrease in the number of endogenous stem cells in cartilage is regarded as the cause of cartilage degeneration. Kartogenin (KGN) is known to induce chondrogenesis of cartilage stem/progenitor cells (CSPCs). Using CSPCs isolated from rat cartilage, we analysed changes in the transcriptome after treatment with KGN in vitro. An animal model of destabilization of the medial meniscus (DMM) was then used to identify the effect of intra-articular (IA) KGN injection on CSPC proliferation in vivo. Here, we demonstrated that KGN promoted the proliferation of CSPCs isolated from cartilage. The percentage of G2-M phase cells in the KGN-treated group reached over 10%, nearly twice that in the control group. Transcriptomic profiling of rat CSPCs revealed significant changes in KGN-treated samples compared to control samples. The gene expression levels of IL-6 and its coreceptor Gp130 were much higher in the KGN-treated group than in the control group. Phosphorylation of the IL-6 downstream molecule Stat3 was enhanced via KGN stimulation. The DMM animal model showed increased articular cartilage thickness after IA KGN injection. IHC staining also demonstrated upregulation of Stat3 phosphorylation and enhanced distribution of CD44+/CD105+ cells in cartilage following IA KGN injection. Thus, our data suggested that KGN promoted cartilage regeneration at least partially by stimulating IL-6/Stat3-dependent proliferation.

Feeder-free generation and transcriptome characterization of functional mesenchymal stromal cells from human pluripotent stem cells.

Induced mesenchymal stromal cells (iMSCs) derived from human pluripotent stem cells (PSCs) are attractive cells for regenerative medicine. However, the transcriptome of iMSCs and signature genes that can distinguish MSCs from fibroblasts and other cell types are rarely explored. In this study, we reported an optimized feeder-free method for the generation of iMSCs from human pluripotent stem cells. These iMSCs display a typical MSC morphology, express classic MSC markers (CD29, CD44, CD73, CD90, CD105, CD166), are negative for lymphocyte markers (CD11b, CD14, CD31, CD34, CD45, HLA-DR), and are potent for osteogenic and chondrogenic differentiation. Using genome-wide transcriptome profiling, we created an easily accessible transcriptome reference for the process of differentiating PSCs into iMSCs. The iMSC transcriptome reference revealed clear patterns in the silencing of pluripotency genes, activation of lineage commitment genes, and activation of mesenchymal genes during iMSC generation. All previously known positive and negative markers for MSCs were confirmed by our iMSC transcriptomic reference, and most importantly, gene classification and time course analysis identified 52 genes including FN1, TGFB1, TAGLN and SERPINE1, which showed significantly higher expression in MSCs (over 3 folds) than fibroblasts and other cell types. Taken together, these results provide a useful method and important resources for developing and understanding iMSCs in regenerative medicine.

Donor genetic backgrounds contribute to the functional heterogeneity of stem cells and clinical outcomes.

Stable and sustainable stem cell sources for stem cell-based therapies are scarce and a key bottleneck for clinical applications. The regenerative potential of stem cells is usually attributed to several allogeneic or even autologous donor-related factors. Genetic background and epigenetic variations in different individuals may significantly affect the functional heterogeneity of stem cells. Particularly, single-nucleotide polymorphisms (SNPs) have been implicated in diseases with monogenetic or multifactorial and complex genetic etiologies. However, the possible effects of individual SNPs on donor stem cells remain far from fully elucidated. In this Perspective, we will discuss the roles played by donor genetic traits in the functional heterogeneity of induced pluripotent stem cells, mesenchymal stem cells, and hematopoietic stem cells and their implications for regenerative medicine and therapy.

RSK-3 promotes cartilage regeneration via interacting with rpS6 in cartilage stem/progenitor cells.

Rationale: Cartilage stem/progenitor cells (CSPC) are a promising cellular source to promote endogenous cartilage regeneration in osteoarthritis (OA). Our previous work indicates that ribosomal s6 kinase 3 (RSK-3) is a target of 4-aminobiphenyl, a chemical enhancing CSPC-mediated cartilage repair in OA. However, the primary function and mechanism of RSK-3 in CSPC-mediated cartilage pathobiology remain undefined. Methods: We systematically assessed the association of RSK-3 with OA in three mouse strains with varying susceptibility to OA (MRL/MpJ>CBA>STR/Ort), and also RSK-3-/- mice. Bioinformatic analysis was used to identify the possible mechanism of RSK-3 affecting CSPC, which was further verified in OA mice and CSPC with varying RSK-3 expression induced by chemicals or gene modification. Results: We demonstrated that the level of RSK-3 in cartilage was positively correlated with cartilage repair capacities in three mouse strains (MRL/MpJ>CBA>STR/Ort). Enhanced RSK-3 expression by 4-aminobiphenyl markedly attenuated cartilage injury in OA mice and inhibition or deficiency of RSK-3 expression, on the other hand, significantly aggravated cartilage damage. Transcriptional profiling of CSPC from mice suggested the potential role of RSK-3 in modulating cell proliferation. It was further shown that the in vivo and in vitro manipulation of the RSK-3 expression indeed affected the CSPC proliferation. Mechanistically, ribosomal protein S6 (rpS6) was activated by RSK-3 to accelerate CSPC growth. Conclusion: RSK-3 is identified as a key regulator to enhance cartilage repair, at least partly by regulating the functionality of the cartilage-resident stem/progenitor cells.

Kartogenin hydrolysis product 4-aminobiphenyl distributes to cartilage and mediates cartilage regeneration.

Rationale The small molecule Kartogenin (KGN) promotes cartilage regeneration in osteoarthritis (OA) by activating stem cells differentiation, but its pharmacological mode-of-action remains unclear. KGN can be cleaved into 4-aminobiphenyl (4-ABP) and phthalic acid (PA) following enzymolysis of an amide bond. Therefore, this study investigated whether 4-ABP or PA exerted the same action as KGN. Methods KGN, 4-ABP and PA were analyzed in cartilage of mice after oral, intravenous or intra-articular administration of KGN by liquid chromatography-mass spectrometry method. Their effect on proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) was evaluated in vitro. Furthermore, their effect on cartilage preservation was tested in mice OA model induced by destabilization of medial meniscus. OA severity was quantified using OARSI histological scoring. Transcriptional analysis was used to find the possible targets of the chemicals, which were further validated. Results We demonstrated that while oral or intra-articular KGN delivery effectively ameliorated OA phenotypes in mice, only 4-ABP was detectable in cartilage. 4-ABP could induce chondrogenic differentiation and proliferation of MSC in vitro and promote cartilage repair in OA mouse models mainly by increasing the number of CD44+/CD105+ stem-cell and prevention of matrix loss. These effect of 4-ABP was stronger than that of KGN. Transcriptional profiling of 4-ABP-stimulated MSC suggested that RPS6KA2 and the PI3K-Akt pathway were 4-ABP targets; 4-ABP could activate the PI3K-Akt pathway to promote MSC proliferation and repair OA injury, which was blocked in RPS6KA2-knockdown MSC or RPS6KA2-deficient mice.Conclusion 4-ABP bio-distribution in cartilage promotes proliferation and chondrogenic differentiation of MSC, and repairs osteoarthritic lesions via PI3K-Akt pathway activation.

One-step Derivation of Functional Mesenchymal Stem Cells from Human Pluripotent Stem Cells.

Mesenchymal stem cells (MSCs) are invaluable cell sources for understanding stem cell biology and potential application in tissue engineering and regenerative medicine. The current issues of MSCs that demand to be further addressed are limited donors, tissue sources and limited capacity of ex vivo expansion. Here, we describe a simple and easy protocol for generating functional mesenchymal stem cells from human pluripotent stem cells (hPSCs) via one-step low glucose medium switch strategy in feeder-free culture system. In this protocol, human induced pluripotent stem cells (hiPSCs) and H9 human embryonic stem cells (hESCs) were successfully differentiated into MSCs, named hiPSC-MSCs and hESC-MSCs, respectively. The derived hiPSC-MSCs and hESC-MSCs exhibited common MSC characteristics as MSCs derived from human bone marrow (hBM-MSCs), including expressing MSC surface markers and possessing capability of tri-lineage differentiation in vitro (adipogenesis, osteogenesis and chondrogenesis). As compared with other available protocols, our protocol can be applied to generate a large number of MSCs from hPSCs with high efficiency, low-cost manner, moreover, not involving embryoid body, mouse feeder-cell, flow sorting, and pathway inhibitors (such as SB203580 and SB431542). We believe that this protocol could provide a robust platform to reach the future demand for producing the industrial scale of MSC from hPSCs for autologous cell-based therapy.

Upregulation of miR­598 promotes cell proliferation and cell cycle progression in human colorectal carcinoma by suppressing INPP5E expression.

Colorectal cancer (CRC) is one of the most common types of cancer worldwide. Recently, microRNAs (miRs) have been considered as novel therapeutic targets for the treatment of cancer. miR­598 is a poorly investigated miR. The underlying mechanism of miR­598 in CRC cells remains to be elucidated. In the present study, miR­598 was demonstrated to be significantly upregulated in CRC tissue by analyzing data from The Cancer Genome Atlas and the Gene Expression Omnibus. The results of a polymerase chain reaction demonstrated that miR­598 expression was significantly upregulated in CRC tissues and cells. Gain of function and loss of function assays demonstrated that miR­598 significantly promoted cell proliferation and cell cycle progression. miR­598 was demonstrated to modulate cell functions by regulating 72 kDa inositol polyphosphate­5­phosphatase (INPP5E). In addition, knockdown of INPP5E counteracted the growth arrest caused by an miR­598­inhibitor. In conclusion, the present study demonstrated that miR­598 contributed to cell proliferation and cell cycle progression in CRC by targeting INPP5E.