Increased In Vitro Osteopotential in SHED Associated with Higher IGF2 Expression When Compared with hASCs

Abstract

Mesenchymal stem cell (MSC) osteogenic differentiation potential varies according to factors such as tissue source and cell population heterogeneity. Pre-selection of cell subpopulations harboring higher osteopotential is a promising strategy to achieve a thorough translation of MSC-based therapies to the clinic. Here, we searched for novel molecular markers predictive of osteopotential by comparing MSC populations from two sources harboring different osteogenic potentials. We show that MSCs from human deciduous teeth (SHED) have an intrinsically higher osteogenic potential when compared with MSCs from human adipose tissue (hASCs) under the same in vitro controlled induction system. Transcriptome profiling revealed IGF2 to be one of the top upregulated transcripts before and during early in vitro osteogenic differentiation. Further, exogenous IGF2 supplementation enhanced alkaline phosphatase activity and matrix mineralization, and inhibition of IGF2 lessened these parameters in SHED and hASCs, validating IGF2 as an osteogenic factor in these MSCs. Further, we found IGF2 to be biallelically expressed in SHED, but not in hASCs. We observed a 4 % methylation increase in the imprinting control region within the IGF2-H19 locus in SHED, and this is mainly due to 2 specific CpG sites. Thus, we suggest that IGF2 upregulation in SHED is due to loss of imprinting. This study unravels osteogenic properties in SHED, implying IGF2 as a potential biomarker of MSCs with higher osteopotential, and unveils IGF2 loss-of-imprinting in SHED.

The Roles of Insulin-Like Growth Factors in Mesenchymal Stem Cell Niche

“…In dental pulp MSC differentiation, IGF-1 was shown to promote mTOR signaling pathway in order to trigger the expression of RUNX2, OCN, OSX, and COL1 [36]. Also, human deciduous teeth MSCs with high osteopotential express and secrete IGF-2 that is required for differentiation and mineralization [107]. Overall, IGF-1 and IGF-2 play a significant role in MSC osteogenic differentiation and bone health.…”

Alternative Strategies for Stem Cell Osteogenic Differentiation

“…Bone marrow is considered one of the main sources of MSCs, both in experimental studies and clinical use [26]. Yet, bone marrow MSCs are obtained through a painful surgical incision that produces a low number of harvested cells [28], with only about 0.001-0.01% of the total population of nucleated cells being identified as MSCs [5,29].…”

Biocompósitos eletrofiados de PLLA com alto conteúdo de partículas de fosfatos de cálcio funcionalizados para regeneração óssea

“…A coloração com vermelho de alisarina (ARS) foi utilizada para caracterizar as mineralizações das SHED e MC3T3-E1 que foram cultivadas nas malhas eletrofiadas de PLLA e PLLA/CaP-L por 21 dias. O corante vermelho de alizarina se liga aos sais de cálcio seletivamente e é amplamente utilizado para quantificação da mineralização [73,86,112] . submetidas às mesmas condições de cultura e igualmente coradas por ARS) [87] .…”

“…Estudos mostram que, utilizando-se fases mais solúveis do que a HA, como BCP (fosfato de cálcio bifásico) por exemplo, aumentase a supersaturação de íons cálcio e fosfato no microambiente aumentando a mineralização [27] . Yamada S. et al [128] As SHED foram escolhidas pois possuem elevado potencial de diferenciação e similaridade com células osteoprogenitoras [86] . Esse tipo celular já foi utilizado por outros pesquisadores para reparar defeitos cranianos e reconstruir osso trabecular em camundongos [129,130] .…”

“…Após a adição dos meios osteogênicos, os biomateriais fibrosos foram recolhidos nos dias 10, 14 e 21 e ensaio para medir a atividade da fosfatase alcalina (ALP). As células foram tratadas com fosfato de p-nitrofenila, e o p-nitrofenol resultante foi quantificado colorimetricamente a 405 nm[86] .A coloração vermelha de alizarina (ARS) também foi utilizada para caracterizar a formação de mineralização de MC3T3-E1 e SHED nas superfícies dos biocompósitos durante 14 e 21 dias. As malhas eletrofiadas foram lavadas 3 vezes com PBS, fixadas com metanol por 30 minutos e incubadas com vermelho de alizarina (1%, pH 4,2) durante 5 min, após isso, as malhas foram submetidas a sucessivas lavagens com água para remoção do corante em excesso.…”