Self-Assembling Scaffolds Supported Long-Term Growth of Human Primed Embryonic Stem Cells and Upregulated Core and Naïve Pluripotent Markers

The maintenance and expansion of human embryonic stem cells (ESCs) in two-dimensional (2-D) culture is technically challenging, requiring routine manipulation and passaging. We developed three-dimensional (3-D) scaffolds to mimic the in vivo microenvironment for stem cell proliferation. The scaffolds were made of two 8-arm polyethylene glycol (PEG) polymers functionalized with thiol (PEG-8-SH) and acrylate (PEG-8-Acr) end groups, which self-assembled via a Michael addition reaction. When primed ESCs (H9 cells) were mixed with PEG polymers, they were encapsulated and grew for an extended period, while maintaining their viability, self-renewal, and differentiation potential both in vitro and in vivo. Three-dimensional (3-D) self-assembling scaffold-grown cells displayed an upregulation of core pluripotency genes, OCT4, NANOG, and SOX2. In addition, the expression of primed markers decreased, while the expression of naïve markers substantially increased. Interestingly, the expression of mechanosensitive genes, YAP and TAZ, was also upregulated. YAP inhibition by Verteporfin abrogated the increased expression of YAP/TAZ as well as core and naïve pluripotent markers. Evidently, the 3-D culture conditions induced the upregulation of makers associated with a naïve state of pluripotency in the primed cells. Overall, our 3-D culture system supported the expansion of a homogenous population of ESCs and should be helpful in advancing their use for cell therapy and regenerative medicine

Transcriptomic Analysis of Naïve Human Embryonic Stem Cells Cultured in Three-Dimensional PEG Scaffolds

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 the 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 the 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

Regulation of self-renewal and senescence in primitive mesenchymal stem cells by Wnt and TGFβ signaling

Abstract Background The therapeutic application of multipotent mesenchymal stem cells (MSCs) encounters significant challenges, primarily stemming from their inadequate growth and limited self-renewal capabilities. Additionally, as MSCs are propagated, their ability to self-renew declines, and the exact cellular and molecular changes responsible for this are poorly understood. This study aims to uncover the complex molecular mechanisms that govern the self-renewal of primitive (p) MSCs. Methods We grew pMSCs using two types of medium, fetal bovine serum (FM) and xeno-free (XM), at both low passage (LP, P3) and high passage (HP, P20). To evaluate LP and HP pMSCs, we examined their physical characteristics, cell surface markers, growth rate, colony-forming ability, BrdU assays for proliferation, telomerase activity, and potential to differentiate into three lineages. Moreover, we conducted RNA-seq to analyze their transcriptome and MNase-seq analysis to investigate nucleosome occupancies. Results When grown in FM, pMSCs underwent changes in their cellular morphology, becoming larger and elongated. This was accompanied by a decrease in the expression of CD90 and CD49f, as well as a reduction in CFE, proliferation rate, and telomerase activity. In addition, these cells showed an increased tendency to differentiate into the adipogenic lineage. However, when grown in XM, pMSCs maintained their self-renewal capacity and ability to differentiate into multiple lineages while preserving their fibroblastoid morphology. Transcriptomic analysis showed an upregulation of genes associated with self-renewal, cell cycle regulation, and DNA replication in XM-cultured pMSCs, while senescence-related genes were upregulated in FM-cultured cells. Further analysis demonstrated differential nucleosomal occupancies in self-renewal and senescence-related genes for pMSCs grown in XM and FM, respectively. These findings were confirmed by qRT-PCR analysis, which revealed alterations in the expression of genes related to self-renewal, cell cycle regulation, DNA replication, differentiation, and senescence. To understand the underlying mechanisms, we investigated the involvement of Wnt and TGFβ signaling pathways by modulating them with agonists and antagonists. This experimental manipulation led to the upregulation and downregulation of self-renewal genes in pMSCs, providing further insights into the signaling pathways governing the self-renewal and senescence of pMSCs. Conclusion Our study shows that the self-renewal potential of pMSCs is associated with the Wnt pathway, while senescence is linked to TGFβ

DataSheet1_The potential role of integrin alpha 6 in human mesenchymal stem cells.docx

Human mesenchymal stem cells (MSCs) are isolated from various adult and perinatal tissues. Although mesenchymal stem cells from multiple sources exhibit similar morphology and cell surface markers, they differ in their properties. In this study, we determined that the expression of integrin alpha 6 (ITGA6) and ITGA6 antisense RNA (ITGA6-AS1) correlates with the proliferation, cell size, and differentiation potential. The expression of ITGA6 was inversely correlated with ITGA6-AS1 in MSCs. The expression of ITGA6 was higher, but ITGA6-AS1 was lower in MSCs from cord placenta junction, cord tissue, and Wharton’s jelly. In contrast, ITGA6 expression was lower, while ITGA6-AS1 was higher in MSCs from the placenta. The bioinformatic analysis showed that ITGA6 genomic DNA transcribes ITGA6-AS1 from the reverse strand, overlapping ITGA6 exon-2. Additionally, we identify several putative promoters (P1-P10) of ITGA6. ITGA6-P10 is CG rich and contains CGI. EMBOSS Cpgplot software revealed a CGI length of 180 bp that extends from nucleotide 125 to 304 of the P10 sequence. We suggest that the post-transcriptional regulation of the ITGA6 in mesenchymal stem cells is controlled by the ITGA6-AS1, which could be a critical factor responsible for the heterogeneity in function and cell fate of human MSCs. These results may provide further impetus for investigations to unravel the mechanisms of ITGA6 regulation that could help maintain or improve the properties of mesenchymal stem cells.</p

Table4_The potential role of integrin alpha 6 in human mesenchymal stem cells.DOCX

Human mesenchymal stem cells (MSCs) are isolated from various adult and perinatal tissues. Although mesenchymal stem cells from multiple sources exhibit similar morphology and cell surface markers, they differ in their properties. In this study, we determined that the expression of integrin alpha 6 (ITGA6) and ITGA6 antisense RNA (ITGA6-AS1) correlates with the proliferation, cell size, and differentiation potential. The expression of ITGA6 was inversely correlated with ITGA6-AS1 in MSCs. The expression of ITGA6 was higher, but ITGA6-AS1 was lower in MSCs from cord placenta junction, cord tissue, and Wharton’s jelly. In contrast, ITGA6 expression was lower, while ITGA6-AS1 was higher in MSCs from the placenta. The bioinformatic analysis showed that ITGA6 genomic DNA transcribes ITGA6-AS1 from the reverse strand, overlapping ITGA6 exon-2. Additionally, we identify several putative promoters (P1-P10) of ITGA6. ITGA6-P10 is CG rich and contains CGI. EMBOSS Cpgplot software revealed a CGI length of 180 bp that extends from nucleotide 125 to 304 of the P10 sequence. We suggest that the post-transcriptional regulation of the ITGA6 in mesenchymal stem cells is controlled by the ITGA6-AS1, which could be a critical factor responsible for the heterogeneity in function and cell fate of human MSCs. These results may provide further impetus for investigations to unravel the mechanisms of ITGA6 regulation that could help maintain or improve the properties of mesenchymal stem cells.</p

Potential of Human Nucleus Pulposus-Like Cells Derived From Umbilical Cord to Treat Degenerative Disc Disease

Degenerative disc disease (DDD) is a common spinal disorder that manifests with neck and lower back pain caused by the degeneration of intervertebral discs (IVDs). Currently, there is no treatment to cure this debilitating ailment. To investigate the potential of nucleus pulposus (NP)-like cells (NPCs) derived from human umbilical cord mesenchymal stem cells (MSCs) to restore degenerated IVDs using a rabbit DDD model. NPCs differentiated from MSCs were characterized using quantitative real-time reverse transcription polymerase chain reaction and immunocytochemical analysis. MSCs and NPCs were labeled with fluorescent dye, PKH26, and transplanted into degenerated IVDs of a rabbit model of DDD (n = 9 each). Magnetic resonance imaging of the IVDs was performed before and after IVD degeneration, and following cell transplantation. IVDs were extracted 8 wk post-transplantation and analyzed by various biochemical, immunohistological, and molecular techniques. NPC derivatives of MSCs expressed known NP-specific genes, SOX9, ACAN, COL2, FOXF1, and KRT19. Transplanted cells survived, dispersed, and integrated into the degenerated IVDs. IVDs augmented with NPCs showed significant improvement in the histology, cellularity, sulfated glycosaminoglycan and water contents of the NP. In addition, expression of human genes, SOX9, ACAN, COL2, FOXF1, KRT19, PAX6, CA12, and COMP, as well as proteins, SOX9, ACAN, COL2, and FOXF1, suggest NP biosynthesis due to transplantation of NPCs. Based on these results, a molecular mechanism for NP regeneration was proposed. The findings of this study demonstrating feasibility and efficacy of NPCs to regenerate NP should spur interest for clinical studies to treat DDD using cell therapy