Human Mesenchymal Stem Cells Retain Multilineage Differentiation Capacity Including Neural Marker Expression after Extended In Vitro Expansion

Abstract

The suitability of human mesenchymal stem cells (hMSCs) in regenerative medicine relies on retention of their proliferative expansion potential in conjunction with the ability to differentiate toward multiple lineages. Successful utilisation of these cells in clinical applications linked to tissue regeneration requires consideration of biomarker expression, time in culture and donor age, as well as their ability to differentiate towards mesenchymal (bone, cartilage, fat) or non-mesenchymal (e.g., neural) lineages. To identify potential therapeutic suitability we examined hMSCs after extended expansion including morphological changes, potency (stemness) and multilineage potential. Commercially available hMSC populations were expanded in vitro for > 20 passages, equating to > 60 days and > 50 population doublings. Distinct growth phases (A-C) were observed during serial passaging and cells were characterised for stemness and lineage markers at representative stages (Phase A: P+5, approximately 13 days in culture; Phase B: P+7, approximately 20 days in culture; and Phase C: P+13, approximately 43 days in culture). Cell surface markers, stem cell markers and lineage-specific markers were characterised by FACS, ICC and Q-PCR revealing MSCs maintained their multilineage potential, including neural lineages throughout expansion. Co-expression of multiple lineage markers along with continued CD45 expression in MSCs did not affect completion of osteogenic and adipogenic specification or the formation of neurospheres. Improved standardised isolation and characterisation of MSCs may facilitate the identification of biomarkers to improve therapeutic efficacy to ensure increased reproducibility and routine production of MSCs for therapeutic applications including neural repair.

Characterization and Comparison of Human and Ovine Mesenchymal Stromal Cells from Three Corresponding Sources

“…However, on day 21 of induction, mRNA expression of Runx2 was up-regulated in all MSCs from both human and ovine sources compared to the corresponding controls as well as compared to day 1 of induction ( Figure 7A,B, top panels). The second osteogenic lineage-specific gene, Col1A, showed no significant change on day 1, but was clearly down-regulated on day 21 in both MSCs from human and ovine sources compared to the corresponding controls ( Figure 7A,B, bottom panels), suggesting a feedback down-regulation as has been described previously both at mRNA [23] and protein level [24].…”

Human bone marrow‐derived MSCs spontaneously express specific Schwann cell markers

“…Our results demonstrate that undifferentiated hBM‐MSCs were already pre‐disposed to neural phenotypes or spontaneously express some neural and specific glial markers without any introduction of inducing agents. Similar findings were observed as reported elsewhere (Tondreau et al, ; Blondheim et al, ; Keilhoff et al, ; Barnabé et al, ; Foudah et al, ; Moradi et al, ; Heng et al, ; Schuh et al, ; Okolicsanyi et al, ). Ravasi et al (), in an in vitro myelination assay (co‐culturing of MSCs with DRG neurons), have provided compelling evidence whereby undifferentiated MSC completely myelinated the neurons with an increasing degree of complexity and maturity throughout the weeks in the co‐culture without the need for Schwann cells transdifferentiation cocktails.…”

“…Furthermore, the guidelines may need to address variation arising from species differences, tissue source, age, gender, and culture conditions, as these may hamper reliable analyses (Uccelli et al, ; Siegel et al, ; Okolicsanyi et al, ) and limit standardisation of cultures and differentiation protocols (Ladak et al, ). Besides, the listed markers also fail to distinguish between MSCs (derived from different tissues) and fibroblasts following mono‐layer expansion; thus, their specificity is questionable (Bara et al, ; Lv et al, ).…”

Bone morphogenetic protein 9 induces osteogenic differentiation of germ cell 1 spermatogonial cells

“…GC-1spg cells are largely involved in spermatogenesis and have mesenchymal stem cell (MSC) differentiation potential [5,6,7,8]. In other words, GC-1spg cells can differentiate into different cell types, including those of the osteogenic, chondrogenic, or adipogenic lineages [9,10,11,12]. However, several molecular events [13,14] and factors are involved in osteogenic differentiation of MSCs.…”