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Matrix reverses immortalization-mediated stem cell fate determination.

ABSTRACT: Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins as top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of the matrix protein influence on surrounding stem cell differentiation.

SUBMITTER: Wang Y

PROVIDER: S-EPMC7944411 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Matrix reverses immortalization-mediated stem cell fate determination

Project description:Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40 LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. In the study, we found that SV40 LT transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by passage 5 IPFSCs yielded IPFSCs with dramatically increased proliferation and chondrogenic differentiation capacity; however, this enhanced capacity diminished if IPFSCs were grown on dECM deposited by passage 15 IPFSCs. Interestingly, expansion on dECM deposited by SV40 LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40 LT transduced passage 15 cells. Our immunofluorescence staining and proteomics data identify matrix components such as basement membrane proteins top candidates for matrix mediated IPFSC rejuvenation. Both cell proliferation and differentiation were endorsed by transcripts measured by RNASeq during the process. This study provides a promising model for in-depth investigation of matrix protein influence on surrounding stem cell differentiation.

2022-02-16 | PXD021069 | Pride

Matrix reverses immortalization-mediated stem cell fate determination

Project description:Primary cell culture in vitro suffers from cellular senescence. We hypothesized that expansion on decellularized extracellular matrix (dECM) deposited by simian virus 40 large T antigen (SV40 LT) transduced autologous infrapatellar fat pad stem cells (IPFSCs) could rejuvenate high-passage IPFSCs in both proliferation and chondrogenic differentiation. Passage 1 (P1) IPFSCs (CTR group) were transduced with lentivirus carrying either SV40 LT (SV40 group) or GFP (GFP group). P5, P10 and P15 cells from each group were compared in proliferation, chondrogenic and adipogenic capacities. P15 IPFSCs were expanded on tissue culture flasks or dECMs deposited by P5 (“young”) or P15 (“old”) IPFSCs with (SP5 and SP15, respectively) or without transduction of SV40 LT (CP5 and CP15, respectively). Cell proliferation was evaluated using population doubling time, flow cytometry for EdU incorporation, cell cycle and surface markers, and real-time quantitative PCR (qPCR) for stemness and senescence gene expression. Chondrogenic capacity was evaluated using Alcian blue staining for sulfated GAGs, immunostaining for type II collagen and qPCR for chondrogenic markers. Adipogenic capacity was evaluated using Oil Red O staining for lipid droplets, western blot and qPCR for adipogenic markers. Proteomics analysis was used to evaluate matrix components from dECMs, expanded cells and chondrogenically induced pellets. We found that SV40 transduced IPFSCs exhibited increased proliferation and adipogenic potential but decreased chondrogenic potential. Expansion on dECMs deposited by SV40 LT transduced IPFSCs yielded IPFSCs with enhanced proliferation and chondrogenic capacity but decreased adipogenic potential, particularly for the dECM group derived from SV40 LT transduced P15 cells. This study provides a promising model for in-depth investigation of ECM proteins to determine how these matrix proteins affect surrounding stem cells’ differentiation preference.

2020-10-04 | GSE148632 | GEO

Matrix reverses immortalization-mediated stem cell fate determination

Project description:Matrix reverses immortalization-mediated stem cell fate determination

| PRJNA625241 | ENA

A matrix micropatterning platform for cell localization and stem cell fate determination.

Project description:To study the role of cell-extracellular matrix (ECM) interactions, microscale approaches provide the potential to perform high throughput assessment of the effect of the ECM microenvironment on cellular function and phenotype. Using a microscale direct writing (MDW) technique, we characterized the generation of multicomponent ECM microarrays for cellular micropatterning, localization and stem cell fate determination. ECMs and other biomolecules of various geometries and sizes were printed onto epoxide-modified glass substrates to evaluate cell attachment by human endothelial cells. The endothelial cells displayed strong preferential attachment to the ECM patterned regions and aligned their cytoskeleton along the direction of the micropatterns. We next generated ECM microarrays that contained one or more ECM components (namely gelatin, collagen IV and fibronectin) and then cultured murine embryonic stem cell (ESCs) on the microarrays. The ESCs selectively attached to the micropatterned features and expressed markers associated with a pluripotent phenotype, such as E-cadherin and alkaline phosphatase, when maintained in growth medium containing leukemia inhibitory factor. In the presence of the soluble factors retinoic acid and bone morphogenetic protein-4 the ESCs differentiated towards the ectodermal lineage on the ECM microarray with differential ECM effects. The ESCs cultured on gelatin showed significantly higher levels of pan cytokeratin expression, when compared with cells cultured on collagen IV or fibronectin, suggesting that gelatin preferentially promotes ectodermal differentiation. In summary, our results demonstrate that MDW is a versatile approach to print ECMs of diverse geometries and compositions onto surfaces, and it is amenable to the generation of multicomponent ECM microarrays for stem cell fate determination.

| S-EPMC2957527 | biostudies-literature

Bi-directional cell-pericellular matrix interactions direct stem cell fate.

Project description:Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells' 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells' reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC's interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.

| S-EPMC6170409 | biostudies-literature

Cell fate determination factor Dachshund reprograms breast cancer stem cell function.

Project description:The cell fate determination factor Dachshund was cloned as a dominant inhibitor of the hyperactive epidermal growth factor receptor ellipse. The expression of Dachshund is lost in human breast cancer associated with poor prognosis. Breast tumor-initiating cells (TIC) may contribute to tumor progression and therapy resistance. Here, endogenous DACH1 was reduced in breast cancer cell lines with high expression of TIC markers and in patient samples of the basal breast cancer phenotype. Re-expression of DACH1 reduced new tumor formation in serial transplantations in vivo, reduced mammosphere formation, and reduced the proportion of CD44(high)/CD24(low) breast tumor cells. Conversely, lentiviral shRNA to DACH1 increased the breast (B)TIC population. Genome-wide expression studies of mammary tumors demonstrated DACH1 repressed a molecular signature associated with stem cells (SOX2, Nanog, and KLF4) and genome-wide ChIP-seq analysis identified DACH1 binding to the promoter of the Nanog, KLF4, and Lin28 genes. KLF4/c-Myc and Oct4/Sox2 antagonized DACH1 repression of BTIC. Mechanistic studies demonstrated DACH1 directly repressed the Nanog and Sox2 promoters via a conserved domain. Endogenous DACH1 regulates BTIC in vitro and in vivo.

| S-EPMC3023510 | biostudies-literature

Immobilization after injury alters extracellular matrix and stem cell fate.

Project description:Cells sense the extracellular environment and mechanical stimuli and translate these signals into intracellular responses through mechanotransduction, which alters cell maintenance, proliferation, and differentiation. Here we use a mouse model of trauma-induced heterotopic ossification (HO) to examine how cell-extrinsic forces impact mesenchymal progenitor cell (MPC) fate. After injury, single-cell (sc) RNA sequencing of the injury site reveals an early increase in MPC genes associated with pathways of cell adhesion and ECM-receptor interactions, and MPC trajectories to cartilage and bone. Immunostaining uncovers active mechanotransduction after injury with increased focal adhesion kinase signaling and nuclear translocation of transcriptional coactivator TAZ, inhibition of which mitigates HO. Similarly, joint immobilization decreases mechanotransductive signaling, and completely inhibits HO. Joint immobilization decreases collagen alignment and increases adipogenesis. Further, scRNA sequencing of the HO site after injury with or without immobilization identifies gene signatures in mobile MPCs correlating with osteogenesis, and signatures from immobile MPCs with adipogenesis. scATAC-seq in these same MPCs confirm that in mobile MPCs, chromatin regions around osteogenic genes are open, whereas in immobile MPCs, regions around adipogenic genes are open. Together these data suggest that joint immobilization after injury results in decreased ECM alignment, altered MPC mechanotransduction, and changes in genomic architecture favoring adipogenesis over osteogenesis, resulting in decreased formation of HO.

| S-EPMC7524473 | biostudies-literature

Biomaterials for pluripotent stem cell engineering: From fate determination to vascularization.

Project description:Recent advancements in material science and engineering may hold the key to overcoming reproducibility and scalability limitations currently hindering the clinical translation of stem cell therapies. Biomaterial assisted differentiation commitment of stem cells and modulation of their in vivo function could have significant impact in stem cell-centred regenerative medicine approaches and next gen technological platforms. Synthetic biomaterials are of particular interest as they provide a consistent, chemically defined, and tunable way of mimicking the physical and chemical properties of the natural tissue or cell environment. Combining emerging biomaterial and biofabrication advancements may finally give researchers the tools to modulate spatiotemporal complexity and engineer more hierarchically complex, physiologically relevant tissue mimics. In this review we highlight recent research advancements in biomaterial assisted pluripotent stem cell (PSC) expansion and three dimensional (3D) tissue formation strategies. Furthermore, since vascularization is a major challenge affecting the in vivo function of engineered tissues, we discuss recent developments in vascularization strategies and assess their ability to produce perfusable and functional vasculature that can be integrated with the host tissue.

| S-EPMC4948647 | biostudies-literature

Author Correction: Bi-directional cell-pericellular matrix interactions direct stem cell fate.

Project description:In the original version of this Article the dataset identifier in the Data Availability statement was incorrect. The correct dataset identifier is PXD009500. This has been corrected in the HTML and PDF versions of this Article.

| S-EPMC6235857 | biostudies-literature

Mettl5 mediated 18S rRNA N6-methyladenosine (m<sup>6</sup>A) modification controls stem cell fate determination and neural function.

Project description:Ribosome RNA (rRNA) accounts for more than 80% of the cell's total RNA, while the physiological functions of rRNA modifications are poorly understood. Mutations of 18S rRNA m6A methyltransferase METTL5 cause intellectual disability, microcephaly, and facial dysmorphisms in patients, however, little is known about the underlying mechanisms. In this study, we identified METTL5 protein complex and revealed that METTL5 mainly interacts with RNA binding proteins and ribosome proteins. Functionally, we found that Mettl5 knockout in mESCs leads to the abnormal craniofacial and nervous development. Moreover, using Mettl5 knockout mouse model, we further demonstrated that Mettl5 knockout mice exhibit intellectual disability, recapitulating the human phenotype. Mechanistically, we found that Mettl5 maintains brain function and intelligence by regulating the myelination process. Our study uncovered the causal correlation between mis-regulated 18S rRNA m6A modification and neural function defects, supporting the important physiological functions of rRNA modifications in human diseases.

| S-EPMC8720661 | biostudies-literature

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