A comparative study of mesenchymal stem cells cultured as cell-only aggregates and in encapsulated hydrogels.
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ABSTRACT:
SUBMITTER: Passanha FR
PROVIDER: S-EPMC9297862 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Physical and cognitive disabilities are hallmarks of a variety of neurological diseases. Stem cell-based therapies are promising solutions to neuroprotect and repair the injured brain and overcome the limited capacity of the central nervous system to recover from damage. It is widely accepted that most benefits of different exogenously transplanted stem cells rely on the secretion of different factors and biomolecules that modulate inflammation, cell death and repair processes in the damaged host tissue. However, few cells survive in cerebral tissue after transplantation, diminishing the therapeutic efficacy. As general rule, cell encapsulation in natural and artificial polymers increases the in vivo engraftment of the transplanted cells. However, we have ignored the consequences of such encapsulation on the secretory activity of these cells. In this study, we investigated the biological compatibility between silk fibroin hydrogels and stem cells of mesenchymal origin, a cell population that has gained increasing attention and popularity in regenerative medicine. Although the survival of mesenchymal stem cells was not affected inside hydrogels, this biomaterial format caused adhesion and proliferation deficits and impaired secretion of several angiogenic, chemoattractant and neurogenic factors while concurrently potentiating the anti-inflammatory capacity of this cell population through a massive release of TGF-Beta-1. Our results set a milestone for the exploration of engineering polymers to modulate the secretory activity of stem cell-based therapies for neurological disorders.
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Project description:Despite recent advances in tissue engineered heart valves (TEHV), a major challenge is identifying a cell source for seeding TEHV scaffolds. Native heart valves are durable because valve interstitial cells (VICs) maintain tissue homeostasis by synthesizing and remodeling the extracellular matrix. This study demonstrates that induced pluripotent stem cells (iPSC)-derived mesenchymal stem cells (iMSCs) can be derived from iPSCs using a feeder-free protocol and then further matured into VICs by encapsulation within 3D hydrogels. The differentiation efficiency was characterized using flow cytometry, immunohistochemistry staining, and trilineage differentiation. Using our feeder-free differentiation protocol, iMSCs were differentiated from iPSCs and had CD90+, CD44+, CD71+, αSMA+, and CD45- expression. Furthermore, iMSCs underwent trilineage differentiation when cultured in induction media for 21 days. iMSCs were then encapsulated in poly(ethylene glycol)diacrylate (PEGDA) hydrogels grafted with adhesion peptide (RGDS) to promote remodeling and further maturation into VIC-like cells. VIC phenotype was assessed by the expression of alpha-smooth muscle actin (αSMA), vimentin, and collagen production after 28 days. When MSC-derived cells were encapsulated in PEGDA hydrogels that mimic the leaflet modulus, a decrease in αSMA expression and increase in vimentin was observed. In addition, iMSCs synthesized collagen type I after 28 days in 3D hydrogel culture. Thus, the results from this study suggest that iMSCs may be a promising cell source for TEHV.Statement of significanceDeveloping a suitable cell source is a critical component for the success and durability of tissue engineered heart valves. The significance of this study is the generation of iPSCs-derived mesenchymal stem cells (iMSCs) that have the capacity to mature into valve interstitial-like cells when introduced into a 3D cell culture designed to mimic the layers of the valve leaflet. iMSCs were generated using a feeder-free protocol, which is one major advantage over other methods, as it is more clinically relevant. In addition to generating a potential new cell source for heart valve tissue engineering, this study also highlights the importance of a 3D culture environment to influence cell phenotype and function.
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Project description:Synthetic hydrogel scaffolds that can be used as culture systems that mimic the natural stem cell niche are of increased importance for stem cell biology and regenerative medicine. These artificial niches can be utilized to control the stem cell fate and will have potential applications for expanding/differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. In this study, we synthesized hyaluronic acid (HA) hydrogels that could be degraded through a combination of cell-released enzymes and used them to culture mouse mesenchymal stem cells (mMSC). To form the hydrogels, HA was modified to contain acrylate groups and crosslinked through Michael addition chemistry using non-degradable, plasmin degradable or matrix metalloproteinase (MMP) degradable crosslinkers. Using this hydrogel we found that mMSC proliferation occurred in the absence of cell spreading, that mMSCs could only spread when both RGD and MMP degradation sites were present in the hydrogel and that mMSCs in hydrogels with high density of RGD (1000 ?m) spread and migrated faster and more extensively than in hydrogels with low density of RGD (100 ?m).
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Project description:BackgroundThin endometrium is a primary cause of defective endometrial receptivity, resulting in infertility or recurrent miscarriage. Much effort has been devoted toward regenerating thin endometrium by stem cell-based therapies. The human placenta-derived mesenchymal stem cells (HP-MSCs) are emerging alternative sources of MSCs with various advantages. To maximize their retention inside the uterus, we loaded HP-MSCs with cross-linked hyaluronic acid hydrogel (HA hydrogel) to investigate their therapeutic efficacy and possible underlying mechanisms.MethodsEthanol was injected into the mice uterus to establish the endometrium-injured model. The retention time of HP-MSCs and HA hydrogel was detected by in vivo imaging, while the distribution of HP-MSCs was detected by immunofluorescence staining. Functional restoration of the uterus was assessed by testing embryo implantation rates. The endometrial morphological alteration was observed by H&E staining, Masson staining, and immunohistochemistry. In vitro studies were further conducted using EdU, transwell, tube formation, and western blot assays.ResultsInstilled HP-MSCs with HA hydrogel (HP-MSCs-HA) exhibited a prolonged retention time in mouse uteri than normal HP-MSCs. In vivo studies showed that the HP-MSCs-HA could significantly increase the gland number and endometrial thickness (P < 0.001, P < 0.05), decrease fibrous area (P < 0.0001), and promote the proliferation and angiogenesis of endometrial cells (as indicated by Ki67 and VEGF, P < 0.05, P < 0.05, respectively) in mice injured endometrium. HP-MSCs-HA could also significantly improve the embryo implantation rate (P < 0.01) compared with the ethanol group. Further mechanistic study showed the paracrine effects of HP-MSCs. They could not only promote the proliferation and migration of human endometrial stromal cells via the JNK/Erk1/2-Stat3-VEGF pathway but also facilitate the proliferation of glandular cells via Jak2-Stat5 and c-Fos-VEGF pathway. In turn, the increased VEGF in the endometrium promoted the angiogenesis of endothelial cells.ConclusionOur study suggested the potential therapeutic effects and the underlying mechanisms of HP-MSCs-HA on treating thin endometrium. HA hydrogel could be a preferable delivery method for HP-MSCs, and the strategy represents a promising therapeutic approach against endometrial injury in clinical settings.
| S-EPMC8822809 | biostudies-literature
Project description:Cell-matrix and cell-biomolecule interactions play critical roles in a diversity of biological events including cell adhesion, growth, differentiation, and apoptosis. Evidence suggests that a concise crosstalk of these environmental factors may be required to direct stem cell differentiation toward matured cell type and function. However, the culmination of these complex interactions to direct stem cells into highly specific phenotypes in vitro is still widely unknown, particularly in the context of implantable biomaterials. In this study, we utilized tunable hydrogels based on a simple high pressure CO2 method and silk fibroin (SF) the structural protein of Bombyx mori silk fibers. Modification of SF protein starting water solution concentration results in hydrogels of variable stiffness while retaining key structural parameters such as matrix pore size and β-sheet crystallinity. To further resolve the complex crosstalk of chemical signals with matrix properties, we chose to investigate the role of 3D hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Our data revealed the potential to upregulate matured vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Overall, our observations suggest that chemical and physical stimuli within the cellular microenvironment are tightly coupled systems involved in the fate decisions of hMSCs. The production of tunable scaffold materials that are biocompatible and further specialized to mimic tissue-specific niche environments will be of considerable value to future tissue engineering platforms.Statement of significanceThis article investigates the role of silk fibroin hydrogel stiffness and transforming growth factor (TGF-β1), with the aim of correlating the effects on the vascular commitment of human mesenchymal stem cells. Specifically, we demonstrate the upregulation of mature vascular smooth muscle cell phenotype (myosin heavy chain expression) of hMSCs by employing appropriate matrix stiffness and growth factor (within 72h). Moreover, we demonstrate the potential to direct specialized hMSC differentiation by modulating stiffness and growth factor using silk fibroin, a well-tolerated and -defined biomaterial with an impressive portfolio of tissue engineering applications. Altogether, our study reinforce the fact that complex differentiation protocols may be simplified by engineering the cellular microenvironment on multiple scales, i.e. matrix stiffness with growth factor.
| S-EPMC4728007 | biostudies-literature
Project description:Angiogenesis has been recognized to play an essential role in remodeling new bone (osteogenesis). Small extracellular vesicles (sEVs), the endogenously secreted nanovesicles by cells, exhibit great potential in the regeneration of bone defects and the realization of cell-free therapy. Chitosan, a natural polysaccharide, can form a thermosensitive injectable hydrogel through the addition of β-glycerophosphate. Herein, we developed injectable thermosensitive hydrogel-encapsulated sEVs derived from bone mesenchymal stem cells, which significantly prolonged delivery and release and synergistically enhanced bone regeneration. sEVs were isolated and characterized, and the physicochemical properties, release kinetics, and biocompatibility of the hydrogels were analyzed. In vitro experiments were performed to investigate osteogenic differentiation, cell proliferation and migration, and tube formation. Thereafter, sEVs were added to the chitosan/β-glycerophosphate hydrogel (sEV@CS/β-GP composite) to repair calvarial defects in rats. The results showed that sEV-loaded hydrogels were biocompatible, exhibiting excellent thermosensitive properties and enhancing bone regeneration. Furthermore, mechanistic studies revealed that exosomal miR-21 targeted SPRY2, thereby promoting angiogenesis. Our study provides new insights on the repair of bone defects with multifunctional controlled-sEV-release hydrogels, which shows great potential in the repair of tissues in the future.
| S-EPMC8807520 | biostudies-literature
Project description:Adipose-derived mesenchymal stem cells (ADSCs) show considerable promise for clinical applications in regenerative medicine. We performed a large-scale single-cell transcriptomic sequencing of 24,358 cultured human ADSCs from three donors. We provide a high-quality dataset, which would be a valuable resource for dissecting the intrapopulation heterogeneity of cultured ADSCs as well as interrogating lineage priming patterns for any interested lineages at single-cell resolution.
| S-EPMC6390702 | biostudies-literature
Project description:Generation of induced pluripotent stem cells (iPSCs) and their differentiation into mesenchymal stem/stromal cells (iMSCs) have created exciting source of cells for autologous therapy. In this study, we have compared the therapeutic potential of iMSCs generated from urinary epithelial (UE) cells with the available umbilical cord MSCs (UC-MSCs). For this, adult UE cells were treated with the mRNA of pluripotent genes (OCT4, NANOG, SOX2, KLF4, MYC and LIN28) and a cocktail of miRNAs under specific culture conditions for generating iPSCs. Our non-viral and mRNA-based treatment regimen demonstrated a high reprogramming efficiency to about 30% at passage 0. These UE-iPSCs were successfully differentiated further into ectoderm, endoderm and mesoderm lineage of cells. Moreover, these UE-iPSCs were subsequently differentiated into iMSCs and were compared with the UC-MSCs. These iMSCs were capable of differentiating into osteocytes, chondrocytes and adipocytes. Our qRT-PCR and Western blot data showed that the CD73, CD90 and CD105 gene transcripts and proteins were highly expressed in iMSCs and UC-MSCs but not in other cells. The comparative qRT-PCR data showed that the iMSCs maintained their MSC characteristics without any chromosomal abnormalities even at later passages (P15), during which the UC-MSCs started losing their MSC characteristics. Importantly, the wound-healing property demonstrated through migration assay was superior in iMSCs when compared to the UC-MSCs. In this study, we have demonstrated an excellent non-invasive and pain-free method of obtaining iMSCs for regenerative therapy. These homogeneous autologous highly proliferative iMSCs may provide an alternative source of cells to UC-MSCs for treating various diseases.
| S-EPMC8435459 | biostudies-literature
Project description:It is difficult to achieve minimally invasive injectable cell delivery while maintaining high cell retention and animal survival for in vivo stem cell therapy of myocardial infarction. Here we show that pluripotent stem cell aggregates pre-differentiated into the early cardiac lineage and encapsulated in a biocompatible and biodegradable micromatrix, are suitable for injectable delivery. This method significantly improves the survival of the injected cells by more than six-fold compared with the conventional practice of injecting single cells, and effectively prevents teratoma formation. Moreover, this method significantly enhances cardiac function and survival of animals after myocardial infarction, as a result of a localized immunosuppression effect of the micromatrix and the in situ cardiac regeneration by the injected cells.
| S-EPMC5095349 | biostudies-literature
Project description:BackgroundAlthough mesenchymal stem/stromal cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the cell agglomerate, we inquired if the initial number of cells forming the aggregate (aggregate cell number (ACN)) can impact chondrogenesis.MethodsAggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-β1). Mechanical properties of the cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence.ResultsReducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to β-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of cells within aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a β-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting aggregates within collagenous matrix not only decreases the necessity for high cell numbers but also leads to marked improvement in the quality of the deposited tissue.ConclusionsThis study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in cell mechanics coincident with MSC chondrogenesis with potential translational applications.
| S-EPMC6329065 | biostudies-literature