Anin vitroexpansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells

Abstract

Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number.

Into the eyes of bone marrow-derived mesenchymal stem cells therapy for myocardial infarction and other diseases

“…Firstly, no standard method has been putted forward about isolation, purification and specific marker molecules for the identification of BM-MSCs. Secondly, the efficiency of BM-MSCs differentiation is not ideal, which is currently one of the research focus on how to induce BM-MSCs to differentiate to the single specific tissue and cells (46)(47)(48)(49). Thirdly, the signal transduction mechanism and the molecular basis of BM-MSCs' differentiation such as which transcription factors or gene were activated to make it for some specific differentiation is still not clear though it is generally considered to be related to reprogramming of BM-MSCs.…”

Concise Review: Optimizing Expansion of Bone Marrow Mesenchymal Stem/Stromal Cells for Clinical Applications

“…Cryopreservation [16], density gradient isolation [52], and seeding density [48] do not yield appreciable effects on MSC multilineage potential. Despite some disparities [14,15], osteogenic potential is the most commonly retained lineage after passage 5 [13, 16-18, 48, 53] ( Table 1). This collective finding across several studies using different isolation protocols, culture media, growth factor supplements, and expansion seeding density supports the theory that osteogenesis is the default differentiation pathway for MSCs [18].…”

“…Thus, the demand for a fast and reliable ex vivo expansion method is necessary. The 2D paradigm exposes many weaknesses in preserving the inherent robustness of MSCs [13][14][15][16][17][18], even during extremely short in vitro culture durations [13]. MSCs have been genetically modified to overexpress human telomerase reverse transcriptase to combat telomere shortening and overcome the onset of senescence in culture [96].…”

“…Since their discovery in 1976 by Friedenstein et al [12], MSCs have been almost exclusively expanded in vitro on tissue culture plastic (TCP) formed from processed polystyrene. Given that classic in vitro culture is distinctly different from the bone marrow niche from which the cells were extracted, it is not surprising that MSC progenitor properties deteriorate as a function of twodimensional (2D) expansion [13][14][15][16][17][18]. Classic monolayer culture notably lacks the three-dimensional (3D) architecture and composition of bone, oxygen tension, mechanical stimuli, and paracrine signaling with other cell types.…”

“…Classic monolayer culture notably lacks the three-dimensional (3D) architecture and composition of bone, oxygen tension, mechanical stimuli, and paracrine signaling with other cell types. Robust proliferation, multilineage potential, and colony-forming efficiency (CFE) can be compromised by 2D culture [13][14][15][16][17][18]. By mimicking the niche, MSC progenitor potency has been enhanced amid growth factors [19][20][21][22][23][24][25][26], in reduced oxygen tension [27][28][29][30][31][32][33][34][35][36][37], and through alternative expansion in 3D [38][39][40][41][42][43][44][45][46].…”