Human mesenchymal stem cells (hMSCs) expanded with and without fibroblast growth factor (FGF) supplementation were compared with respect to their proliferation rate, ability to differentiate along the chondrogenic pathway in vitro, and their gene expression profiles. hMSCs expanded in FGF-supplemented medium were smaller and proliferated more rapidly than hMSCs expanded in control conditions. Chondrogenic cultures made with FGF-treated cells were larger and contain more proteoglycan than those made with control cells. Furthermore, aggregates of FGF-treated cells lacked the collagen type I-positive and collagen type II-negative outer layer characteristic of aggregates of control cells. A total of 358 unique transcripts were differentially expressed in FGF-treated hMSCs. Of these, 150 were upregulated and 208 downregulated. Seventeen percent of these genes affect proliferation. Known genes associated with cellular signaling functions comprised the largest percentage ( approximately 20%) of differentially expressed transcripts. Eighty percent of differentially expressed extracellular matrix-related genes were downregulated. The present findings that FGF-2 enhances proliferation and differentiation of hMSCs adds to a growing body of evidence that cytokines modulate the differentiation potential and, perhaps, the multipotentiality of adult stem cells. With the generation of gene expression profiles of FGF-treated and control cells we have taken the first steps in the elucidation of the molecular mechanism(s) behind these phenomena.
Human involucrin (hINV) is a cornified envelope precursor that is specifically expressed in the suprabasal epidermal layers. We previously demonstrated that 2500 base pairs of the hINV gene upstream regulatory region confers differentiation appropriate regulation in transgenic mice. An analysis of the hINV gene sequence upstream of the transcription start site reveals five potential AP1 binding sites (AP1-1 to 5). Using reporter gene constructs in human keratinocytes, we show that the most distal (AP1-5) and most proximal (AP1-1) AP1 sites are essential for high level transcriptional activity. Simultaneous mutation of these sites reduces transcription by 80%. Gel supershift experiments indicate the interaction of these sites with Fra-1, junB, and junD. Involucrin mRNA levels increase 10-fold and promoter activity 5-11-fold when differentiation is induced by phorbol ester. Functional studies implicate AP1-1 and AP1-5 in mediating the phorbol ester-dependent increase in promoter activity. No involucrin promoter activity or involucrin mRNA was detected in 3T3 fibroblasts. We conclude that (i) two AP1 sites in the hINV promoter are important elements required for keratinocyte-specific expression, (ii) these AP1-1 sites mediate the phorbol ester-dependent increase in promoter activity, and (iii) Fra-1, junB, and junD may be important regulators of hINV expression in epidermis.
An accurate, rapid, and cost‐effective biosensor for the quantification of disease biomarkers is vital for the development of early‐diagnostic point‐of‐care systems. The recent discovery of the trans‐cleavage property of CRISPR type V effectors makes CRISPR a potential high‐accuracy bio‐recognition tool. Herein, a CRISPR‐Cas12a (cpf1) based electrochemical biosensor (E‐CRISPR) is reported, which is more cost‐effective and portable than optical‐transduction‐based biosensors. Through optimizing the in vitro trans‐cleavage activity of Cas12a, E‐CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV‐16) and parvovirus B19 (PB‐19), with a picomolar sensitivity. An aptamer‐based E‐CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF‐β1) protein in clinical samples. As demonstrated, E‐CRISPR could enable the development of portable, accurate, and cost‐effective point‐of‐care diagnostic systems.
Articular cartilage repair and regeneration provides a substantial challenge in Regenerative Medicine because of the high degree of morphological and mechanical complexity intrinsic to hyaline cartilage due, in part, to its extracellular matrix. Cartilage remains one of the most difficult tissues to heal; even state-of-the-art regenerative medicine technology cannot yet provide authentic cartilage resurfacing. Mesenchymal stem cells (MSCs) were once believed to be the panacea for cartilage repair and regeneration, but despite years of research, they have not fulfilled these expectations. It has been observed that MSCs have an intrinsic differentiation program reminiscent of endochondral bone formation, which they follow after exposure to specific reagents as a part of current differentiation protocols. Efforts have been made to avoid the resulting hypertrophic fate of MSCs; however, so far, none of these has recreated a fully functional articular hyaline cartilage without chondrocytes exhibiting a hypertrophic phenotype. We reviewed the current literature in an attempt to understand why MSCs have failed to regenerate articular cartilage. The challenges that must be overcome before MSC-based tissue engineering can become a front-line technology for successful articular cartilage regeneration are highlighted.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.