The effect of hypoxia on chondrogenesis of equine synovial membrane-derived and bone marrow-derived mesenchymal stem cells

Abstract

Background Joint injury is extremely common in equine athletes and post-traumatic osteoarthritis (PTOA), a progressive and debilitating disease, is estimated to affect 60% of horses in the USA. The limited potential for intrinsic healing of articular cartilage has prompted intense efforts to identify a cell-based repair strategy to prevent progression of PTOA. Mesenchymal stem cells (MSCs) have the potential to become an ideal source for cell-based treatment of cartilage lesions; however, full chondrogenic differentiation remains elusive. Due to the relatively low oxygen tension in articular cartilage, hypoxia has been proposed as a method of increasing MSC chondrogenesis. The objective of this study was to investigate the effect of hypoxic culture conditions on chondrogenesis in equine synovial membrane-derived MSCs (SM-MSCs) and bone marrow-derived MSCs (BM-MSCs). MSCs were isolated from synovial membrane and bone marrow collected from 5 horses. Flow cytometric analysis was used to assess cell surface marker expression including CD29, CD44, CD90, CD105, CD45, CD-79α, MHCI and MHCII. MSC pellets were cultured in normoxic (21% O 2 ) or in hypoxic (5% O 2 ) culture conditions for 28 days. Following the culture period, chondrogenesis was assessed by histology, biochemical analyses and gene expression of chondrogenic-related genes including ACAN , COL2b , SOX9 , and COL10A1 . Results Both cell types expressed markers consistent with stemness including CD29, CD44, CD90, CD105, and MHCI and were negative for exclusion markers (CD45, CD79α, and MHCII). Although the majority of outcome variables of chondrogenic differentiation were not significantly different between cell types or culture conditions, COL10A1 expression, a marker of chondrocyte hypertrophy, was lowest in hypoxic SM-MSCs and was significantly lower in hypoxic SM-MSCs compared to hypoxic BM-MSCs. Conclusions Hypoxic culture conditions do not appear to increase chondrogenesis of equine SM-MSCs or BM-MSCs; however, hypoxia may downregulate the hypertrophic marker COL10A1 in SM-MSCs.

Low-Intensity Pulsed Ultrasound Alleviates Hypoxia-Induced Chondrocyte Damage in Temporomandibular Disorders by Modulating the Hypoxia-Inducible Factor Pathway

“…Type II collagen expression is positively related with chondrogenic capacity (Gale et al, 2019) and was previously shown to significantly decrease under hypoxic conditions (Anderson et al, 2016). Similarly, we found that hypoxia significantly decreased the expression of type II collagen (Col2) protein in mandibular chondrocytes compared to that in controls and cells treated with LIPUS (Figures 2A, B).…”

Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal Stem Cells

“…Gale et al analyzed the chondrogenic differentiation potential of equine synovial membrane and bone marrow derived MSCs and found no appreciable difference between cells cultured either at 5% oxygen or in normoxic conditions for 28 days. The results of expression of chondrogenic genes SOX9, ACAN, and COL2b were also variable between the groups [79]. Similar to this Li J and Pei M found no significant differences in chondrogenic index between normoxic and 5% hypoxic culturing for 7 days in synovium derived MSCs [80].…”

Therapeutic Use of Mesenchymal Stromal Cells: The Need for Inclusive Characterization Guidelines to Accommodate All Tissue Sources and Species

“…In a review of MSCs derived from other species, all species noted some difficulties exhibiting expression of the 3 classic MSC markers. In the equine literature, CD73 and CD105 are most often unreported or negative (de Mattos Carvalho et al, 2009 ; Radcliffe et al, 2010 ; Ranera et al, 2011 ; Maia et al, 2013 ; Barberini et al, 2014 ; Alipour et al, 2015 ; Zahedi et al, 2017 ; Gale et al, 2019 ; Kamm et al, 2019 ; Lepage et al, 2019 ). In mouse literature, there are several examples of researchers being able to demonstrate one marker and not the other two, but no clear pattern as to which marker is shown to have positive expression (Meirelles Lda and Nardi, 2003 ; Anderson et al, 2013 ; Hosseinzadeh Shirzeily et al, 2013 ; Deng et al, 2014 ; Li and Niyibizi, 2016 ; Ahmed et al, 2017 ).…”

“…CD29, integrin beta-1, is a cell surface receptor that is involved in cell adhesion. CD29 has been found to be “highly” expressed (≥95%) on MSCs derived from human (Hu et al, 2003 ; Le Blanc et al, 2003 ; Wexler et al, 2003 ; Brooke et al, 2008 ; Pruszak et al, 2009 ; Park and Patel, 2010 ; Aldridge et al, 2012 ; Al-Nbaheen et al, 2013 ; Guan et al, 2014 ; Yang et al, 2014 ; Alrefaei et al, 2015 ; Katsiani et al, 2016 ; Van Pham et al, 2016 ; Togarrati et al, 2018 ; Kaviani et al, 2019 ), rat (Wu et al, 2009 ; Walker et al, 2010 ; Song et al, 2014 ; Davies et al, 2015 ; Suto et al, 2017 ), equine (Ranera et al, 2011 ; Alipour et al, 2015 ; Esteves et al, 2017 ; Zahedi et al, 2017 ; Gale et al, 2019 ; Lepage et al, 2019 ), canine (Filioli Uranio et al, 2011 ; Choi et al, 2013 ; Ivanovska et al, 2017 ), mouse (Meirelles Lda and Nardi, 2003 ; Ahmed et al, 2017 ), porcine (Ock et al, 2010 ; Lee et al, 2015 ; Wiater et al, 2018 ), buffalo (Deng et al, 2018 ), rabbit (Lee et al, 2013 ; Kovac et al, 2017 ), bovine (Corradetti et al, 2013 ; de Moraes et al, 2016 ), and chickens (Bai et al, 2013 ). Evidence suggests that CD29 expression may be involved with MSC migration along with CD73 (Ode et al, 2011 ).…”