Co-growth of Stem Cells With Target Tissue Culture as an Easy and Effective Method of Directed Differentiation.

Marina Valentinovna Kovina,Yury Mikhailovich Khodarovich,Sergey Alexandrovich Yakovenko,Tatyana Gennadievna Dyuzheva,Mikhail Evgenievich Krasheninnikov

doi:10.3389/fbioe.2021.591775

Abstract

The long-term co-culture of mouse embryonic stem cells (mESC) with rat endothelial cells (EC) was tested for contact differentiation into the endothelial lineage. Serial passaging of rat ECs mixed with mESC in ratio 10:1 resulted in the emergence of a homogeneous cell population expressing mouse endothelial surface markers CD102, CD29, CD31. Rat endothelial surface marker RECA-1 completely disappeared from the co-cultured population after 2 months of weekly passaging. Co-incubation of mESC with rat ECs without cell-to-cell contact did not result in the conversion of mESC into ECs. After co-cultivation of adult mesenchymal stem cells from human endometrium (eMSC) with pre-hepatocyte-like cells of human hepatocarcinoma Huh7 the resulting co-culture expressed mature liver markers (oval cell antigen and cytokeratin 7), none of which were expressed by any of co-cultivated cultures, thus proving that even an immature (proliferating) pre-hepatocyte-like line can induce hepatic differentiation of stem cells. In conclusion, we have developed conditions where long-term co-proliferation of embryonic or adult SC with fully or partially differentiated cells results in stem cell progeny expressing markers of target tissue. In the case of endothelial differentiation, the template population quickly disappeared from the resulted culture and the pure endothelial population of stem cell progeny emerged. This approach demonstrates the expected fate of stem cells during various in vivo SC-therapies and also might be used as an effective in vitro differentiation method to develop the pure endothelium and, potentially, other tissue types of desirable genetic background.

Highlights

Cell therapies actively incorporate into modern medicine, for treating rare diseases but, in perspective, for wide-spread diseases such as diabetes mellitus (Memon and Abdelalim, 2020) and for aging treatment (Kovina et al, 2019), considering discoveries of new and productive stem cells (SC) sources: induciblepluripotent SC, endometrial mesenchimal SCEffective Differentiation by Long-Term Co-Culture (Kovina et al, 2018)
In order to investigate the fate of mouse embryonic stem cells (mESC) in long-term co-culture with endothelial cells (EC) we performed co-culture experiments with different initial mESC:EC ratios
We have found that serial passaging of co-cultured mESC and rat EC at initial ratio 1:10 results in homogenous culture expressing mouse endothelial markers and no rat endothelial markers

Summary

Introduction

Effective Differentiation by Long-Term Co-Culture (Kovina et al, 2018). The key issue of successful cell therapy is the effective differentiation of stem cells into target cell type. Co-culture is one of the known differentiation strategies of modern science, in line with the addition of bioactive substances like growth factors, hormones etc. Even if in vivo chimerizm might be sometimes explained without differentiation, but at least in vitro the direct cell-to-cell contact during SC co-cultivation with differentiated cells promotes partial differentiation into myocytes, endotheliocytes, and hepatocytes with little or no fusion (Thiele et al, 2004; Wurmser et al, 2004; Xu et al, 2004; Lange et al, 2005), though no full conversion was yet shown. The cell-contact might work in combination with the secreted factors and growth factors from the medium (Bigdeli et al, 2009; Banerjee et al, 2011; Domev et al, 2012)

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Conclusion