Cartilaginous and osteochondral tissue formation by human mesenchymal stem cells on three-dimensionally woven scaffolds
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ABSTRACT:
PROVIDER: PRJNA488091 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7759644_1.fastq.gz | Fastqsanger.gz | |||
| SRR7759644_2.fastq.gz | Fastqsanger.gz | |||
| SRR7759645_1.fastq.gz | Fastqsanger.gz | |||
| SRR7759645_2.fastq.gz | Fastqsanger.gz | |||
| SRR7759646_1.fastq.gz | Fastqsanger.gz |
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Project description:The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remain important challenges for regenerative medicine. The objective of this study was to assess of adult human mesenchymal stem cells (MSC) in large, three dimensionally woven poly(-caprolactone) (PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations: PCL alone, PCL-bone, “point-of-care” seeded MSC-PCL-bone, and chondrogenically pre-cultured Ch-MSC-PCL-bone constructs were implanted in a dorsal, ectopic pouch in a nude rat. After eight weeks, cells in the PCL scaffold exhibited both chondrogenic and osteogenic gene expression profiles. Notably, while both tissue profiles were present, constructs that had been chondrogenically pre-cultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabeculae-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions, and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.
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Project description:Recently the role of macrophages on osteogenesis of mesenchymal stem cells (MSCs) is brought to focus. On the other hand, the cross-talk between macrophages and MSCs confirms that the paracrine molecules derived from macrophages are also carefully regulated by MSCs. Human umbilical cord mesenchymal stem cells (hucMSCs) could reduce secretions of inflammatory factors from macrophages for improving injury healing. hucMSC-derived extracellular matrix (hucMSC-ECM) exhibit nature similar to hucMSCs. It has been demonstrated that hucMSC-ECM possess considerable effects on reducing inflammatory response of macrophages, while the role of hucMSC-ECM on the expression of macrophage-derived paracrine osteogenic molecules is unclear. Here, we presented the decalcified bone scaffolds modified by hucMSC-derived extracellular matrix (DBM-ECM) maintaining multiple soluble cytokines including macrophage migration inhibitory factor (MIF). Compared with DBM, the DBM-ECM scaffolds induced bone formation in a macrophage-depending manner by an improved heterotopic ossification in SCID mice with or without macrophage depletion. Macrophage cocultured with the DBM-ECM expressed four osteoinductive cytokines (BMP2, FGF2, TGFβ3 and OSM), which were screen out by RNA sequencing, qPCR and western blot. The conditioned medium from macrophages cocultured with the DBM-ECM improved osteogenic differentiation of hBMSCs and activated CD74/CD44 signal transduction including phosphorylation of P38 and dephosphorylation of c-jun. Furthermore, the inhibitory effects of the DBM-ECM scaffolds with knockdown of MIF on osteogenesis in vitro and in vivo revealed that macrophage-mediated osteogenesis depends on MIF/CD74/ P38 signal transduction. This study indicates the coordinated crosstalk of macrophages and MSCs play a key role on bone regeneration induced by the DBM-ECM, with the emphasis on the constructing macrophage-derived osteoimmunological microenvironment.
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Project description:Naïve human embryonic stem cells (ESCs) are characterized by improved viability, proliferation, and differentiation capacity in comparison to traditionally derived primed human ESCs. However, currently used two-dimensional (2-D) cell culture techniques fail to mimic the three-dimensional (3-D) in vivo microenvironment, altering morphological and molecular characteristics of ESCs. Here, we describe the use of 3-D self-assembling scaffolds that support growth and maintenance of the naïve state characteristics of ESC line, Elf1. Scaffolds were formed via a Michael addition reaction upon combination of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups. 3-D scaffold environment maintained the naïve state and supported long-term growth of ESCs. RNA-sequencing demonstrated significant changes in gene expression profiles between 2-D and 3-D grown cells. Gene Ontology analysis revealed upregulation of biological processes involved in the regulation of transcription and translation, extracellular matrix organization, and chromatin remodeling in 3-D grown cells. 3-D culture conditions also induced upregulation of genes associated with Wnt and focal adhesion signaling, while p53 signaling pathway associated genes were downregulated. Our findings for the first time provide insight into the possible mechanisms of self-renewal of naïve ESCs stimulated by the transduction of mechanical signals from the 3-D microenvironment.
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