Fate Of Mesenchymal Stem Cells Research Articles

3D-printable supramolecular hydrogels with shear-thinning property: fabricating strength tunable bioink via dual crosslinking

3-dimensional (3D) bioprinting technology provides promising strategy in the fabrication of artificial tissues and organs. As the fundamental element in bioprinting process, preparation of bioink with ideal mechanical properties without sacrifice of biocompatibility is a great challenge. In this study, a supramolecular hydrogel-based bioink is prepared by polyethylene glycol (PEG) grafted chitosan, α-cyclodextrin (α-CD) and gelatin. It has a primary crosslinking structure through the aggregation of the pseudo-polyrotaxane-like side chains, which are formed from the host-guest interactions between α-CD and PEG side chain. Apparent viscosity measurement shows the shear-shinning property of this bioink, which might be due to the reversibility of the physical crosslinking. Moreover, with β-glycerophosphate at different concentrations as the secondary crosslinking agent, the printed constructs demonstrate different Young's modulus (p < 0.001). They could also maintain the Young's modulus in cell culture condition for at least 21 days (p < 0.05). By co-culturing each component with fibroblasts, CCK-8 assay demonstrate cellular viability is higher than 80%. After bioprinting and culturing, immunofluorescence staining with quantification indicate the expression of Ki-67, Paxillin, and N-cadherin is higher in day 14 than those in day 3 (p < 0.05). Oil red O and Nissl body specific staining reflect strength tunable bioink may have impact on the cell fate of mesenchymal stem cells (p < 0.05). This work might provide new idea for advanced bioink in the application of re-establishing complicated tissues and organs.

Cell culture dimensionality influences mesenchymal stem cell fate through cadherin-2 and cadherin-11

The acquisition of a specific cell fate is one of the core aims of tissue engineering and regenerative medicine. Significant evidence shows that aggregate cultures have a positive influence on cell fate decisions, presumably through cell-cell interactions, but little is known about the specific mechanisms. To investigate the difference between cells cultured as a monolayer and as aggregates, we started by looking at cadherin expression, an important protein involved in cell adhesion, during the differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in aggregate and monolayer cultures. We observed that proliferating hMSCs in monolayer culture expressed lower levels of cadherin-2 and increased cadherin-11 expression at cell-cell contact sites over time, which was not evident in the aggregate cultures. By knocking down cadherin-2 and cadherin-11, we found that both cadherins were required for adipogenic differentiation in a monolayer as well as aggregate culture. However, during osteogenic differentiation, low levels of cadherin-2 were found to be favorable for cells cultured as a monolayer and as aggregates, whereas cadherin- 11 was dispensable for cells cultured as aggregates. Together, these results provide compelling evidence for the important role that cadherins play in regulating the differentiation of hMSCs and how this is affected by the dimensionality of cell culture.

Epigenetic modifier trichostatin A enhanced osteogenic differentiation of mesenchymal stem cells by inhibiting NF-κB (p65) DNA binding and promoted periodontal repair in rats.

We wished to evaluate whether epigenetic modifiers have a beneficial effect on treating experimental periodontitisand mechanisms forregulating the cell fate of mesenchymal stem cells (MSCs) in inflammatory microenvironments. We isolated MSCs from healthy and inflamed gingival tissues to investigatewhether trichostatin A (TSA) could improve osteogenic differentiation and resolve inflammation in vitro. The tissue regenerative potentials were evaluated when treated with a temperature-dependent, chitosan-scaffold-encapsulated TSA, in a rat model of periodontitis. After induction with the conditioned medium, TSA treatment increased the osteogenic differentiation potential of inflamed MSCs and healthy MSCs. In addition, interleukin-6 and interleukin-8 levels in supernatants were significantly decreased after TSA treatment. Moreover, TSA promoted osteogenic differentiation by inhibiting nuclear factor-κB (p65) DNA binding in MSCs. In rats with experimental periodontitis, 7 weeks after local injections of chitosan-scaffold-encapsulated TSA, histology and microcomputed tomography showed a significant increase in alveolar bone volume and less inflammatory infiltration compared with vehicle-treated rats. The concentrations of interferon-γ and interleukin-6 were significantly decreased in the gingival crevicular fluid after TSA treatment. This study demonstrated that TSA had anti-inflammatory properties and could promote periodontal tissue repair, which indicated that epigenetic modifiers hold promise as a potential therapeutic option for periodontal tissue repair.

High-Throughput Screening and Hierarchical Topography-Mediated Neural Differentiation of Mesenchymal Stem Cells.

Biophysical factors such as anisotropic topography composed of micro/nanosized structures are important for directing the fate of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and have been applied to neuronal differentiation. Via high-throughput screening (HTS) methods based on topography gradients, the optimum topography is determined and translated toward a hierarchical architecture designed to mimic the nerve nano/microstructure. The polydimethylsiloxane (PDMS)-based topography gradient with amplitudes (A) from 541 to 3073 nm and wavelengths (W) between 4 and 30 µm is developed and the fate commitment of MSC toward neuron lineage is investigated. The hierarchical structures, combining nano- and microtopography (W0.3/W26 parallel/perpendicular) are fabricated to explore the combined topography effects on neuron differentiation. From the immunofluorescent staining results (Tuj1 and MAP2), the substrate characterized by W: 26 µm; A: 2.9 µm shows highest potential for promoting neurogenesis. Furthermore, the hierarchical features (W0.3/W26 parallel) significantly enhance neural differentiation. The hBM-MSCs on the hierarchical substrates exhibit a significantly lower percentage of nuclear Yes-associated protein (YAP)/TAZ and weaker cell contractility indicating that the promoted neurogenesis is mediated by the cell tension and YAP/TAZ pathway. This research provides new insight into designing biomaterials for applications in neural tissue engineering and contributes to the understanding of topography-mediated neuronal differentiation.

Genetic and epigenetic stability of stem cells: Epigenetic modifiers modulate the fate of mesenchymal stem cells

Stem cell research has progressed widely and has been receiving a considerable attention for its advantages and drawbacks. Despite their extensive therapeutic potential in regenerative medicine, they are debatable for their genetic and epigenetic stability. In fact lineage specific differentiation is mediated via epigenetic changes in DNA methylation, acetylation, histone modifications etc. Thus epigenetics plays an important role in stem cell biology. For therapeutic interventions stem cells need to be genetically and epigenetically stable for their maximum paracrine secretions for bringing about expected tissue repair and regeneration. In this review we have focused on the current status of genetic and epigenetic stability in stem cells and their importance in regenerative medicine. We have also touched upon the possibility of considering tissue resident mesenchymal stem cells as epigenetic modifiers. This is likely to open a new era in stem cell therapeutic intervention by reversing disease inducing epigenetic changes.

Contributions of Dickkopf-1 to Obesity-Induced Bone Loss and Marrow Adiposity.

ABSTRACTLow bone strength in overweight individuals is a significant medical problem. One important determinant of mesenchymal stem cell fate into osteoblasts or adipocytes is the Wnt signaling pathway. We recently showed that Dickkopf‐1 (DKK1), a potent Wnt inhibitor, is upregulated in obese mice. In this study, we investigated the role of DKK1 in the pathogenesis of obesity‐induced bone loss using global and tissue‐specific KO mice. Obesity was induced in 8‐week‐old male mice with an inducible global (Rosa26‐CreERT2) or osteoprogenitor‐ (Osx–Cre‐) specific deletion of Dkk1 with a high‐fat diet (HFD) containing 60% fat. After 12 weeks, body weight, bone volume, bone fat mass, and bone turnover were assessed. Dkk1 fl/fl ;Rosa26‐CreERT2 mice experienced a similar increase in body weight and white fat pads as control mice. A HFD significantly reduced trabecular bone mass and the bone formation rate in Cre‐ mice and Dkk1 fl/fl ;Rosa26‐CreERT2 mice. Interestingly, Dkk1 fl/fl ;Rosa26‐CreERT2 mice were protected from HFD‐induced cortical bone loss. Furthermore, a HFD was associated with increased bone marrow fat in the femur, which was less pronounced in Dkk1 fl/fl ;Rosa26‐CreERT2 mice. Mice with an osteoprogenitor‐specific Dkk1 deletion showed similar results as the global knockout, showing a protection against HFD‐induced cortical bone loss and an accumulation of bone marrow fat, but a similar decrease in trabecular bone volume. In summary, DKK1 appears to contribute distinctly to cortical, but not trabecular bone loss in obesity. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Citrate Mediates Crosstalk between Mitochondria and the Nucleus to Promote Human Mesenchymal Stem Cell In Vitro Osteogenesis.

Citrate, generated in the mitochondria, is a key metabolite that might link metabolism with signaling, chromatin structure and transcription to orchestrate mesenchymal stem cells (MSCs) fate determination. Based on a detailed morphological analysis of 3D reconstruction of mitochondria and nuclei in single cells, we identified contact sites between these organelles that drastically increase in volume and number during the early stage of mesenchymal stem cell differentiation. These contact sites create a microdomain that facilitates exchange of signals from mitochondria to the nucleus. Interestingly, we found that the citrate derived from mitochondria is necessary for osteogenic lineage determination. Indeed, inhibition of the citrate transporter system dramatically affected osteogenesis, reduced citrate levels that could be converted in α-ketoglutarate, and consequently affected epigenetic marker H3K9me3 associated with the osteogenesis differentiation process. These findings highlight that mitochondrial metabolites play key regulatory roles in the MSCs differentiation process. Further in-depth investigation is needed to provide novel therapeutic strategies in the field of regenerative medicine.

Leptin acts on mesenchymal stem cells to promote chemoresistance in osteosarcoma cells.

Leptin signaling influences osteoblastogenesis and modulates the fate of mesenchymal stem cells (MSCs) during bone and cartilage regeneration. Although MSCs abound in the osteosarcoma (OS) microenvironment, and leptin exhibits pro-tumorigenic properties, leptin’s influence on OS progression and chemoresistant signaling in MSCs remains unclear. Using cell viability and apoptosis assays, we showed that medium conditioned by leptin-treated human MSCs promotes cisplatin resistance in cultured human OS cells. Moreover, GFP-LC3 expression and chloroquine treatment experiments showed that this effect is mediated by stimulation of autophagy in OS cells. TGF-β expression in MSCs was upregulated by leptin and suppressed by leptin receptor knockdown. Silencing TGF-β in MSCs also abolished OS cell chemoresistance induced by leptin-conditioned medium. Cisplatin resistance was also induced when leptin-conditioned MSCs were co-injected with MG-63 OS cells to generate subcutaneous xenografts in nude mice. Finally, we observed a significant correlation between autophagy-associated gene expression in OS clinical samples and patient prognosis. We conclude that leptin upregulates TGF-β in MSCs, which promotes autophagy-mediated chemoresistance in OS cells.

Fluorescent conjugated polymer nanovector for in vivo tracking and regulating the fate of stem cells for restoring infarcted myocardium

Stem cell therapy holds great promise for cardiac regeneration. However, the lack of ability to control stem cell fate after in vivo transplantation greatly restricts its therapeutic outcomes. MicroRNA delivery has emerged as a powerful tool to control stem cell fate for enhanced cardiac regeneration. However, the clinical translation of therapy based on gene-transfected stem cells remains challenging, due to the unknown in vivo behaviors of stem cells. Here, we developed a nano-platform (i.e., PFBT@miR-1-Tat NPs) that can achieve triggered release of microRNA-1 to promote cardiac differentiation of mesenchymal stem cells (MSCs), and long-term tracking of transplanted MSCs through bright and ultra-stable fluorescence of conjugated polymer poly(9,9-dioctylfluorene-alt-benzothiadiazole) (PFBT). We found that PFBT@miR-1-Tat NP-treated MSCs significantly restored the infarcted myocardium by promoting stem cell cardiac differentiation and integration with the in situ cardiac tissues. Meanwhile, MSCs without gene delivery improved the infarcted heart functions mainly through a paracrine effect and blood vessel formation. The developed conjugated polymer nanovector should be a powerful tool for manipulating as well as revealing the fate of therapeutic cells in vivo, which is critical for optimizing the therapeutic route of gene and cell combined therapy and therefore for accelerating clinical translation. Statement of significanceThe lack of controllability in stem cell fate and the unclear in vivo cellular behaviors restrict the therapeutic outcomes of stem cell therapy. Herein, we engineered fluorescent conjugated polymer nanoparticles as gene delivery nanovectors with controlled release and high intracellular delivery capability to harness the fate of mesenchymal stem cells (MSCs) in vivo, meanwhile to reveal the cellular mechanism of gene-treated stem cell therapy. As compared with only MSC treatment that improves infarcted myocardium functions through paracrine effect, treatment with conjugated polymer nanovector-treated MSCs significantly restored infarcted myocardium through enhancing MSC cardiac differentiation and integration with the in-situ cardiac tissues. These findings demonstrate that the conjugated polymer nanovector would be a powerful tool in optimizing gene and cell combined therapy.

Interleukin-11 regulates the fate of adipose-derived mesenchymal stem cells via STAT3 signalling pathways.

ObjectiveAdipose‐derived mesenchymal stem cells (ADSCs) offer great promise as cell therapy for ischaemic diseases. Due to their poor survival in the ischaemic environment, the therapeutic efficacy of ADSCs is still relatively low. Interleukin‐11 (IL‐11) has been shown to play a key role in promoting cell proliferation and protecting cells from oxidative stress injury. The aim of this study was to determine whether IL‐11 could improve therapeutic efficacy of ADSCs in ischaemic diseases.Methods and ResultsADSCs were prepared from inguinal subcutaneous adipose tissue and exposed to hypoxic environment. The protein expression of IL‐11 was decreased after hypoxic treatment. In addition, ADSCs viability was increased after IL‐11 treatment under hypoxia. Moreover, IL‐11 enhanced ADSCs viability in a dose‐dependent manner under normoxia. Importantly, IL‐11 promoted ADSCs proliferation and migration and protected ADSCs against hydrogen peroxide‐induced cellular death. Notably, IL‐11 enhanced ADSCs proliferation and migration, also promoted cell survival and apoptosis resistance by STAT3 signalling. In vivo, mice were subjected to limb ischaemia and treated with IL‐11 overexpression ADSCs and control ADSCs. IL‐11 overexpression ADSCs improved perfusion recovery in the ischaemic muscles.ConclusionsWe provide the evidence that IL‐11 promoted ADSCs proliferation, stimulated ADSCs migration and attenuated ADSCs apoptosis by activation of STAT3 signalling. These results suggest that IL‐11 facilitated ADSCs engraftment in ischaemic tissue, thereby enhanced ADSCs therapeutic efficacy.

Overexpression of bone morphogenetic protein 3 (BMP-3) via raav-mediated gene transfer on the osteochondrogenic fate of human bone marrow-derived mesenchymal stem cells

Purpose: Injured adult articular cartilage is not capable of reliably regenerating an original hyaline structure. To improve the intrinsic healing activities of damaged cartilage, administration of genetically modified bone marrow-derived mesenchymal stem cells (MSCs) in cartilage lesions is a potent approach to improve cartilage repair. Here, we explored the potential benefits of overexpressing the bone morphogenetic protein 3 (BMP-3) [1] via clinically relevant recombinant adeno-associated virus (rAAV) vectors upon the commitment of human MSCs (hMSCs) as future therapeutic platforms for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [2]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-hBMP-3 a human BMP-3 sequence (OriGene), both controlled by the CMV-IE promoter/enhancer [2]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty, washed, and the pellets obtained were resuspended in DMEM, 10% FCS, 1% penicillin-streptomycin, and FGF-2 (10 ng/ml) for selection by adherence of progenitor cells and characterization of stem cell surface markers [2]. Cells (passage 2) were transduced with rAAV (rAAV-hBMP-3 or rAAV-lacZ: 40 μl each vector) or let untreated and kept either in monolayer cultures in DMEM, 10% FBS or as high-density aggregate cultures (2 x 105 cells) in chondrogenic medium (DMEM high glucose, ITS+ Premix, 1 mM pyruvate, 37.5 μg/ml ascorbate 2-phosphate, 10-7 M dexamethasone, 10 ng/ml TGF-β) for up to 21 days. Expression of BMP-3 was monitored by immunohistochemistry or by ELISA (R&D Systems) [2]. Histological analyses were performed on fixed monolayer cultures (ALP staining) or on paraffin-embedded sections of the aggregate cultures (5 μμ) (toluidine blue staining) [2]. Total RNA was extracted using the RNeasy Protect Mini kit (Qiagen) and reverse transcription was carried out with the 1st Strand cDNA Synthesis kit (Qiagen). cDNA amplification was performed via SYBR Green real-time RT-PCR [2]. Ct values were obtained for each target gene (SOX9, COL10A1) and GAPDH as a control for normalization, and fold inductions (relative to control samples) were measured using the 2-ΔΔCt method [2]. Each condition was performed in duplicate in three independent experiments. A t-test was employed with p < 0.05 considered statistically significant. Results: Successful BMP-3 overexpression was observed both in hMSC monolayer and aggregate cultures by application of rAAV-hBMP-3 relative to the control conditions (lack of vector delivery, reporter rAAV-lacZ gene transfer) over the whole period of evaluation (Fig. 1). Application of rAAV-hBMP-3 significantly increased the levels of cell proliferation in hMSC monolayer and aggregate cultures relative to the control conditions (up to 1.3- and 1.4 difference, respectively, p ≤ 0.001) (Table 1). Chondrogenic differentiation was evidenced by intense toluidine blue staining in hMSC aggregate cultures after 21 days, especially when the cells were treated with rAAV-hBMP-3 (Fig. 2). Similar results were noted regarding the osteogenic differentiation of the cells over time (Fig. 2). A real-time RT-PCR analysis revealed a reduced hypertrophic differentiation of the cells following rAAV-hBMP-3 transduction relative to the control treatments (up to 5-fold difference in COL10A1 expression, p ≤ 0.001), probably due to increased chondrogenic SOX9 gene expression levels (up to 8-fold difference, p ≤ 0.001) (Fig. 3). Conclusions: Gene transfer of BMP-3 via rAAV directs the osteochondrogenic commitment of hMSCs with a delay in hypertrophic differentiation. These Results demonstrate the possibility of modifying hMSCs by therapeutic BMP-3 gene transfer as a future, potential tool to enhance the repair processes in sites of osteochondral lesions. REFERENCES: [1] Wozney et al., Science 1988, 242:1528; [2] Venkatesan et al., Stem Cell Res Ther 2012, 3:22. ACKNOWLEDGMENTS: Work funded by the Deutsche Arthrose-Hilfe. The authors wish to thank Mary B. Goldring for helpful discussion.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Postnatal fate of donor mesenchymal stem cells after transamniotic stem cell therapy in a healthy model

PurposeWe sought to examine donor mesenchymal stem cell (MSC) fate after birth following transamniotic stem cell therapy (TRASCET) in a healthy model. MethodsLewis rat fetuses (n = 91) were divided into two groups based on the content of volume-matched intraamniotic injections performed on gestational day 17 (term = 21–22 days): either a suspension of amniotic fluid-derived MSCs (afMSCs) labeled with luciferase (n = 38) or acellular luciferase only (n = 53). Infused afMSCs consisted of syngeneic Lewis rat cells phenotyped by flow cytometry. Samples from 14 anatomical sites (heart, lung, brain, liver, spleen, pancreas, bowel, kidney, thyroid, skin, skeletal muscle, thymus, peripheral blood and bone marrow) from survivors were screened for luciferase activity 16 days after birth. Statistical analysis was by logistic regression and the Wald test (p < 0.05). ResultsOverall survival was 32% (29/91). When controlled by the acellular luciferase injections, donor afMSCs were not identified at any anatomical site in any neonate as measured by relative light units (all p > 0.05). Donor afMSC viability was confirmed in term placentas. ConclusionsDonor mesenchymal stem cells are not detectable in the neonate after intraamniotic injection in a normal syngeneic rodent model. This finding suggests that clinical trials of transamniotic stem cell therapy may be amenable to regulatory approval. Level of evidenceN/A (animal and laboratory study).

Characterization of cell signaling, morphology, and differentiation potential of human mesenchymal stem cells based on cell adhesion mechanism

It is essential to characterize the cellular properties of mesenchymal stem cell populations to maintain quality specifications and control in regenerative medicine. Biofunctional materials have been designed as artificial matrices for the stimulation of cell adhesion and specific cellular functions. We have developed recombinant maltose-binding protein (MBP)-fused proteins as artificial adhesion matrices to control human mesenchymal stem cell (hMSC) fate by using an integrin-independent heparin sulfate proteoglycans-mediated cell adhesion. In this study, we characterize cell adhesion-dependent cellular behaviors of human adipose-derived stem cells (hASCs) and human bone marrow stem cells (hBMSCs). We used an MBP-fused basic fibroblast growth factor (MF)-coated surface and fibronectin (FN)-coated surface to restrict and support, respectively, integrin-mediated adhesion. The cells adhered to MF exhibited restricted actin cytoskeleton organization and focal adhesion kinase phosphorylation. The hASCs and hBMSCs exhibited different cytoplasmic projection morphologies on MF. Both hASCs and hBMSCs differentiated more dominantly into osteogenic cells on FN than on MF. In contrast, hASCs differentiated more dominantly into adipogenic cells on MF than on FN, whereas hBMSCs differentiated predominantly into adipogenic cells on FN. The results indicate that hASCs exhibit a competitive differentiation potential (osteogenesis vs. adipogenesis) that depends on the cell adhesion matrix, whereas hBMSCs exhibit both adipogenesis and osteogenesis in integrin-mediated adhesion and thus hBMSCs have noncompetitive differentiation potential. We suggest that comparing differentiation behaviors of hMSCs with the diversity of cell adhesion is an important way to characterize hMSCs for regenerative medicine.

Unidirectional rotating molecular motors dynamically interact with adsorbed proteins to direct the fate of mesenchymal stem cells.

Artificial rotary molecular motors convert energy into controlled motion and drive a system out of equilibrium with molecular precision. The molecular motion is harnessed to mediate the adsorbed protein layer and then ultimately to direct the fate of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). When influenced by the rotary motion of light-driven molecular motors grafted on surfaces, the adsorbed protein layer primes hBM-MSCs to differentiate into osteoblasts, while without rotation, multipotency is better maintained. We have shown that the signaling effects of the molecular motion are mediated by the adsorbed cell-instructing protein layer, influencing the focal adhesion-cytoskeleton actin transduction pathway and regulating the protein and gene expression of hBM-MSCs. This unique molecular-based platform paves the way for implementation of dynamic interfaces for stem cell control and provides an opportunity for novel dynamic biomaterial engineering for clinical applications.

Stiffness-mediated mesenchymal stem cell fate decision in 3D-bioprinted hydrogels.

BackgroundHydrogels with tuneable mechanical properties are an attractive material platform for 3D bioprinting. Thus far, numerous studies have confirmed that the biophysical cues of hydrogels, such as stiffness, are known to have a profound impact on mesenchymal stem cell (MSC) differentiation; however, their differentiation potential within 3D-bioprinted hydrogels is not completely understood. Here, we propose a protocol for the exploration of how the stiffness of alginate–gelatin (Alg-Gel) composite hydrogels (the widely used bioink) affects the differentiation of MSCs in the presence or absence of differentiation inducing factors.MethodsTwo types of Alg-Gel composite hydrogels (Young’s modulus: 50 kPa vs. 225 kPa) were bioprinted independently of porosity. Then, stiffness-induced biases towards adipogenic and osteogenic differentiation of the embedded MSCs were analysed by co-staining with alkaline phosphatase (ALP) and oil red O. The expression of specific markers at the gene level was detected after a 3-day culture.ResultsConfocal microscopy indicated that all tested hydrogels supported MSC growth and viability during the culture period. Higher expression of adipogenic and osteogenic markers (ALP and lipoprotein lipase (LPL)) in stiffer 3D-bioprinted matrices demonstrated a more significant response of MSCs to stiffer hydrogels with respect to differentiation, which was more robust in differentiation-inducing medium. However, the LPL expression in stiffer 3D-bioprinted constructs was reduced at day 3 regardless of the presence of differentiation-inducing factors. Although MSCs embedded in softer hydrogels to some extent proceeded toward adipogenic and osteogenic lineages within a few days, their differentiation seemed to be slower and more limited. Interestingly, the hydrogel itself (without differentiation-inducing factors) exhibited a slight effect on whether MSCs differentiated towards an adipogenic or an osteogenic fate. Considering that the mechano-regulated protein Yes-associated protein (YAP) is involved in MSC fate decisions, we further found that inhibition of YAP significantly downregulated the expression of ALP and LPL in MSCs in stiffer constructs regardless of the induced growth factors present.ConclusionsThese results demonstrate that the differentiation of MSCs in 3D-bioprinted matrices is dependent on hydrogel stiffness, which emphasizes the importance of biophysical cues as a determinant of cellular behaviour.

Small molecules for mesenchymal stem cell fate determination.

Mesenchymal stem cells (MSCs) are adult stem cells harboring self-renewal and multilineage differentiation potential that are capable of differentiating into osteoblasts, adipocytes, or chondrocytes in vitro, and regulating the bone marrow microenvironment and adipose tissue remodeling in vivo. The process of fate determination is initiated by signaling molecules that drive MSCs into a specific lineage. Impairment of MSC fate determination leads to different bone and adipose tissue-related diseases, including aging, osteoporosis, and insulin resistance. Much progress has been made in recent years in discovering small molecules and their underlying mechanisms control the cell fate of MSCs both in vitro and in vivo. In this review, we summarize recent findings in applying small molecules to the trilineage commitment of MSCs, for instance, genistein, medicarpin, and icariin for the osteogenic cell fate commitment; isorhamnetin, risedronate, and arctigenin for pro-adipogenesis; and atractylenolides and dihydroartemisinin for chondrogenic fate determination. We highlight the underlying mechanisms, including direct regulation, epigenetic modification, and post-translational modification of signaling molecules in the AMPK, MAPK, Notch, PI3K/AKT, Hedgehog signaling pathways etc. and discuss the small molecules that are currently being studied in clinical trials. The target-based manipulation of lineage-specific commitment by small molecules offers substantial insights into bone marrow microenvironment regulation, adipose tissue homeostasis, and therapeutic strategies for MSC-related diseases.

Aging aggravates intervertebral disc degeneration by regulating transcription factors toward chondrogenesis.

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.

Vulpinic Acid Controls Stem Cell Fate toward Osteogenesis and Adipogenesis.

Vulpinic acid, a naturally occurring methyl ester of pulvinic acid, has been reported to exert anti-fungal, anti-cancer, and anti-oxidative effects. However, its metabolic action has not been implicated yet. Here, we show that vulpinic acid derived from a mushroom, Pulveroboletus ravenelii controls the cell fate of mesenchymal stem cells and preadipocytes by inducing the acetylation of histone H3 and α-tubulin, respectively. The treatment of 10T1/2 mesenchymal stem cells with vulpinic acid increased the expression of Wnt6, Wnt10a, and Wnt10b, which led to osteogenesis inhibiting the adipogenic lineage commitment, through the upregulation of H3 acetylation. By contrast, treatment with vulpinic acid promoted the terminal differentiation of 3T3-L1 preadipocytes into mature adipocytes. In this process, the increase in acetylated tubulin was accompanied, while acetylated H3 was not altered. As excessive generation of adipocytes occurs, the accumulation of lipid drops was not concentrated, but dispersed into a number of adipocytes. Consistently, the expressions of lipolytic genes were upregulated and inflammatory factors were downregulated in adipocytes exposed to vulpinic acid during adipogenesis. These findings reveal the multiple actions of vulpinic acid in two stages of differentiation, promoting the osteogenesis of mesenchymal stem cells and decreasing hypertrophic adipocytes, which can provide experimental evidence for the novel metabolic advantages of vulpinic acid.

Adaptive Liquid Interfacially Assembled Protein Nanosheets for Guiding Mesenchymal Stem Cell Fate.

There is a growing interest in the development of dynamic adaptive biomaterials for regulation of cellular functions. However, existing materials are limited to two-state switching of the presentation and removal of cell-adhesive bioactive motifs that cannot emulate the native extracellular matrix (ECM) in vivo with continuously adjustable characteristics. Here, tunable adaptive materials composed of a protein monolayer assembled at a liquid-liquid interface are demonstrated, which adapt dynamically to cell traction forces. An ultrastructure transition from protein monolayer to hierarchical fiber occurs through interfacial jamming. Elongated fibronectin fibers promote formation of elongated focal adhesion structures, increase focal adhesion kinase activation, and enhance neuronal differentiation of stem cells. Cell traction force results in spatial rearrangement of ECM proteins, which feeds back to alter stem cell fate. The reported biomimetic adaptive liquid interface enables dynamic control of stem cell behavior and has potential translational applications.

Effects of a Non-Thermal Atmospheric Pressure Plasma Jet with Different Gas Sources and Modes of Treatment on the Fate of Human Mesenchymal Stem Cells

Despite numerous attempts to use human mesenchymal stem cells (hMSCs) in the field of tissue engineering, the control of their differentiation remains challenging. Here, we investigated possible applications of a non-thermal atmospheric pressure plasma jet (NTAPPJ) to control the differentiation of hMSCs. An air- or nitrogen-based NTAPPJ was applied to hMSCs in culture media, either directly or by media treatment in which the cells were plated after the medium was exposed to the NTAPPJ. The durations of exposure were 1, 2, and 4 min, and the control was not exposed to the NTAPPJ. The initial attachment of the cells was assessed by a water-soluble tetrazolium assay, and the gene expression in the cells was assessed through reverse-transcription polymerase chain reaction and immunofluorescence staining. The results showed that the gene expression in the hMSCs was generally increased by the NTAPPJ exposure, but the enhancement was dependent on the conditions of the exposure, such as the source of the gas and the treatment method used. These results were attributed to the chemicals in the extracellular environment and the reactive oxygen species generated by the plasma. Hence, it was concluded that by applying the best conditions for the NTAPPJ exposure of hMSCs, the control of hMSC differentiation was possible, and therefore, exposure to an NTAPPJ is a promising method for tissue engineering.