Prospective Isolation and Characterization of Chondroprogenitors from Human Chondrocytes Based on CD166/CD34/CD146 Surface Markers

Purpose Chondrocytes, isolated from articular cartilage, are routinely utilized in cell-based therapeutics for the treatment of cartilage pathologies. However, restoration of the biological tissue faces hindrance due to the formation of primarily fibrocartilaginous repair tissue. Chondroprogenitors have been reported to display superiority in terms of their chondrogenic potential and lesser proclivity for hypertrophy. In line with our recent results, comparing chondroprogenitors and chondrocytes, we undertook isolation of progenitors from the general pool of chondrocytes, based on surface marker expression, namely, CD166, CD34, and CD146, to eliminate off-target differentiation and generate cells of stronger chondrogenic potential. This study aimed to compare chondrocytes, chondroprogenitors, CD34−CD166+CD146+ sorted chondrocytes, and CD34−CD166+CD146− sorted chondrocytes. Methods Chondrocytes obtained from 3 human osteoarthritic knee joints were subjected to sorting, to isolate CD166+ and CD34− subsets, and then were further sorted to obtain CD146+ and CD146− cells. Chondrocytes and fibronectin adhesion-derived chondroprogenitors served as controls. Assessment parameters included reverse transcriptase polymerase chain reaction for markers of chondrogenesis and hypertrophy, trilineage differentiation, and total GAG/DNA content. Results Based on gene expression analysis, CD34−CD166+CD146+ sorted chondrocytes and chondroprogenitors displayed comparability and significantly higher chondrogenesis with a lower tendency for hypertrophy when compared to chondrocytes and CD34−CD166+CD146− sorted chondrocytes. The findings were also reiterated in multilineage potential differentiation with the 146+ subset and chondroprogenitors displaying lower calcification and chondroprogenitors displaying higher total GAG/DNA content compared to chondrocytes and 146− cells. Conclusion This unique progenitor-like population based on CD34−CD166+CD146+ sorting from chondrocytes exhibits efficient potential for cartilage repair and merits further evaluation for its therapeutic application.

Transcriptional Profiling of Cardiac Cells Links Age-Dependent Changes in Acetyl-CoA Signaling to Chromatin Modifications

Metabolism has emerged as a regulator of core stem cell properties such as proliferation, survival, self-renewal, and multilineage potential. Metabolites serve as secondary messengers, fine-tuning signaling pathways in response to microenvironment alterations. Studies show a role for central metabolite acetyl-CoA in the regulation of chromatin state through changes in histone acetylation. Nevertheless, metabolic regulators of chromatin remodeling in cardiac cells in response to increasing biological age remains unknown. Previously, we identified novel cardiac-derived stem-like cells (CTSCs) that exhibit increased functional properties in the neonatal heart (nCTSC). These cells are linked to a unique metabolism which is altered with CTSC aging (aCTSC). Here, we present an in-depth, RNA-sequencing-based (RNA-Seq) bioinformatic with cluster analysis that details a distinct epigenome present in nCTSCs but not in aCTSCs. Gene Ontology (GO) and pathway enrichment reveal biological processes, including metabolism, gene regulation enriched in nCTSCs, and STRING analysis that identifies a network of genes related to acetyl-CoA that can potentially influence chromatin remodeling. Additional validation by Western blot and qRT-PCR shows increased acetyl-CoA signaling and histone acetylation in nCTSCs compared to aCTSCs. In conclusion, our data reveal that the link between metabolism and histone acetylation in cardiac cells is altered with the aging of the cardiac tissue.

A Non-Enzymatic Method to Obtain a Fat Tissue Derivative Highly Enriched in Adipose Stem Cells (ASCs) from Human Lipoaspirates: Preliminary Results

Adipose tissue possesses phenotypic gene expression characteristics that are similar to human mesenchymal stem cells (hMSCs). Nevertheless, the multilineage potential may be inhibited, and cells may not expand adequately to satisfy the requirements of Good Manufacturing Practice (cGMP). An autologous hMSC-enriched fat product would fulfil the void from a biomedical and clinical perspective. In this study, we suggest a novel mechanism using a closed system without enzymes, additives or other modifications, which will produce non-expanded, accessible material. This decentralized fat product, unlike unprocessed lipoaspirates, adequately encloses the vascular stroma with adipocytes and stromal stalks along with their vascular channels and lumina. This fat product contained hASCs and fewer hematopoietic elements such as lipoaspirates, which were digested enzymatically according to flow cytometric investigations, and molecular analysis also showed significant hASC uniformity within the cells of the stromal vascular tissue. Moreover, the fat product produced a higher quantity of hASCs similar to hMSCs in isolation with the typical characteristics of an osteogenic, chondrogenic and adipogenic lineage. Interestingly, these properties were evident in the non-enzymatic derived adipose tissue, as opposed to hASCs in isolation from the enzymatically digested lipoaspirates, suggesting that the aforementioned procedure may be an adequate alternative to regenerate and engineer tissue for the treatment of various medical conditions and promote efficient patient recovery.

Evaluation of Three Devices for the Isolation of the Stromal Vascular Fraction from Adipose Tissue and for ASC Culture: A Comparative Study

Adipose-derived stem/stromal cells (ASCs) reside in the stromal vascular fraction (SVF) of adipose tissue (AT) and can be easily isolated. However, extraction of the SVF from lipoaspirate is a critical step in generating ASC, and semiautomated devices have been developed to enhance the efficacy and reproducibility of the outcomes and to decrease manipulation and contamination. In this study, we compared the reference method used in our lab for SVF isolation from lipoaspirate, with three medical devices: GID SVF-1™, Puregraft™, and Stem.pras®. Cell yield and their viability were evaluated as well as their phenotype with flow cytometry. Further on, we determined their proliferative potential using population doublings (PD), PD time (PDT), and clonogenicity assay (CFU-F). Finally, we checked their genetic stability using RT-qPCR for TERT mRNA assay and karyotyping as well as their multilineage potential including adipogenic, chondrogenic, and osteogenic differentiation. Our results demonstrate that all the devices allow the production of SVF cells with consistent yield and viability, in less time than the reference method. Expanded cells from the four methods showed no significant differences in terms of phenotype, proliferation capabilities, differentiation abilities, and genetic stability.

Caprine Endometrial Mesenchymal Stromal Stem Cell: Multilineage Potential, Characterization, and Growth Kinetics in Breeding and Anestrous Stages

The endometrial layer of the uterus contains a population of cells with similar characteristics of mesenchymal stem cells (MSCs). In the present study, caprine endometrial mesenchymal stromal stem cells (En-MSCs) characters and differentiation potential to chondrogenic, osteogenic, and adipogenic cell lines as well as their growth kinetics in breeding and anestrous stages were evaluated. En-MSCs were enzymatically isolated from endometrial layer of the uterus of adult goats and were cultured and subcultured until passage 4. The growth kinetics and population doubling time (PDT) of caprine En-MSCs in breeding and anestrous stages were determined. En-MSCs in passage 4 were used for the karyotyping and differentiation into chondrocytes, osteocytes, and adipocytes. The PDT in anestrus phase was 40.6 h and in cyclic goats was 53 h. En-MSCs were fibroblast-like in all passages. The number of chromosomes was normal (2n=60) with no chromosomal instability. Chondrogenic, osteogenic, and adipogenic differentiation of En-MSCs was confirmed by staining with Alcian blue, Alizarin red, and Oil Red O, respectively. Caprine En-MSCs demonstrated to be an alternative source of MSCs for cell therapy purposes in regenerative medicine.