Transcriptomic response of bioengineered human cartilage to parabolic flight microgravity is sex-dependent.

Spaceflight and simulated spaceflight microgravity induced osteoarthritic-like alterations at the transcriptomic and proteomic levels in the articular and meniscal cartilages of rodents. But little is known about the effect of spaceflight or simulated spaceflight microgravity on the transcriptome of tissue-engineered cartilage developed from human cells. In this study, we investigate the effect of simulated spaceflight microgravity facilitated by parabolic flights on tissue-engineered cartilage developed from in vitro chondrogenesis of human bone marrow mesenchymal stem cells obtained from age-matched female and male donors. The successful induction of cartilage-like tissue was confirmed by the expression of well-demonstrated chondrogenic markers. Our bulk transcriptome data via RNA sequencing demonstrated that parabolic flight altered mostly fundamental biological processes, and the modulation of the transcriptome profile showed sex-dependent differences. The secretome profile analysis revealed that two genes (WNT7B and WNT9A) from the Wnt-signaling pathway, which is implicated in osteoarthritis development, were only up-regulated for female donors. The results of this study showed that the engineered cartilage tissues responded to microgravity in a sex-dependent manner, and the reported data offers a strong foundation to further explore the underlying mechanisms.

Mesenchymal stem cell therapy inhibited inflammatory and profibrotic pathways induced by partial bladder outlet obstruction and prevented high-pressure urine storage.

BACKGROUND: Partial bladder outlet obstruction (pBOO) is characterized by an initial inflammatory response that progresses to smooth muscle hypertrophy and fibrosis. Current treatment modalities carry high risk of morbidity. Mesenchymal stem cells (MSCs) are undifferentiated adult cells with reparative, immunomodulatory, and anti-inflammatory capacities. The ability of MSCs to inhibit inflammatory and profibrotic pathways in bladder cells has been recently reported. OBJECTIVES: This study aimed to investigate the therapeutic effects of MSCs on pBOO-induced inflammatory, profibrotic signaling pathways and end-organ physiology. MATERIALS AND METHODS: Twenty Sprague Dawley rats were randomly assigned to 5 groups: unobstructed controls, pBOO for 2 and 4 weeks, pBOO+MSCs for 2 and 4 weeks. Partial bladder outlet obstruction was surgically induced followed by intravenous injection of MSCs. Endpoint urodynamics was performed, and bladder tissue harvested for analysis. Reverse transcription real time polymerase chain reaction (RT-PCR) and immunohistochemistry were performed to study gene and protein expression of major inflammatory and profibrotic markers. RESULTS: Partial bladder outlet obstruction resulted in an upregulation of transforming growth factor beta (TGFß1), mothers against decapentaplegic homolog 2/3 (SMAD2/3), hypoxia inducible factor 1 alpha (HIF1α), hypoxia inducible factor 3 alpha (HIF3α), vascular endothelial growth factor (VEGF), tumor necrosis factor (TNFα), mechanistic target of rapamycin (mTOR), p70 ribosomal S6 protein kinase (p70 S6K), collagen 1 (COL1), and collagen 3 (COL3) expression in a time-dependent manner. This was coupled with a downregulation of interleukin (IL)-10 expression. Increase of bladder fibrosis was directly related to the duration of pBOO and associated with high urine storage pressure. Injected MSCs were identified in the bladder 4 weeks after therapy. The immunomodulatory effect of MSCs(defined by reduced TNFα and increased IL-10 and VEGF) was most predominant 2 weeks after therapy. Significant downregulation of profibrotic genes occurred 4 weeks after therapy. End filling pressure, hypertrophy, and fibrosis were significantly reduced after MSC therapy (P < 0.05). DISCUSSION: Mesenchymal stem cell therapy led to a profound systematic improvement of the obstructed bladder. This included an initial anti-inflammatory response and a subsequent antifibrotic reaction. Essentially, both phases were associated with a reduction of urine storage pressure. The intravenously injected MSCs were tracked in the bladder. However, their presence in non-target organs such as the lungs, spleen, and liver was not tracked. CONCLUSIONS: Partial bladder outlet obstruction induced significant upregulation of hypoxic, inflammatory, and profibrotic markers. Mesenchymal stem cell therapy potently inhibited these pathways and improved bladder function.

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies.

BACKGROUND: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem-cell exhibits tri-lineage differentiation potential and is able to maintain its proliferation potential and cell-surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad-derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. MATERIALS AND METHODS: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell-surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. RESULTS: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell-surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad-derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator-activated receptor γ2 and lipoprotein lipase, and oil red O staining. DISCUSSION: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem-cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.

The epitope characterisation and the osteogenic differentiation potential of human fat pad-derived stem cells is maintained with ageing in later life.

Some clinical settings are deficient in osteogenic progenitors, e.g. atrophic nonunited fractures, large bone defects, and regions of scarring and osteonecrosis. These benefit from the additional use of bone marrow-derived mesenchymal stem cells, but these cells exhibit an age-related decline in lifespan, proliferation and osteogenic potential. Therapeutic approaches for the repair of bone could be optimised by the identification of a stem cell source that does not show age-related changes. Fat pad-derived stem cells are capable of osteogenesis, but a detailed study of the effect of ageing on their epitope profile and osteogenic potential has so far not been performed. Fat pad-derived cells were isolated from 2 groups of 5 patients with a mean age of 57 years (S.D. 3 years) and 86 years (S.D. 3 years). The proliferation, epitope profile and osteogenic differentiation potential of cells from the 2 groups were compared. Cells isolated from the fat pad of both groups showed similar proliferation rates and exhibited a cell surface epitope profile similar but not identical to that of bone marrow-derived stem cells. The cells from both groups cultured in osteogenic medium exhibited osteogenesis as shown by a significant upregulation of alkaline phosphatase and osteocalcin genes, and significantly greater alkaline phosphatase enzyme activity compared to cells cultured in the control medium. The cells cultured in the osteogenic medium also showed greater calcium phosphate deposition on alizarin red staining. There was no significant difference between the osteogenic potential of the two age groups for any of the parameters studied. The fat pad is a consistent and homogenous source of stem cells that exhibits osteogenic differentiation potential with no evidence of any decline with ageing in later life. This has many potential therapeutic tissue engineering applications for the repair of bone defects in an increasingly ageing population.

Nonepitopic antibody binding sequence: implications in screening and development of peptide vaccines.

We describe the interaction of a nonepitopic synthetic decapeptide sequence comprising, GQVLQGAIKG, derived from a random sequence with polyclonal IgGs from various animal sources. GQVLQGAIKG was screened for antibody binding activity using ELISA techniques. The peptide showed similar binding characteristics to the IgGs tested. The results were similar whether we used peptide acid or amide. MAP (multiple antigen peptide)-type construct of the peptide was synthesised and employed as an approach to enhance peptide-IgG interaction. The construct, (GQVLQGAIKG)(4)-K(2)-K, showed significant antibody binding activity relative to its monomeric form. These results show that nonepitopic sequences may contribute to binding activity observed in peptide library screening and development of peptide based vaccines. As a cautionary point the measure of antibody binding cannot alone be used to classify peptide as an antigen.