The molecular mechanisms of growth and expression profiles of proliferating AdMSCs and FBs differentiation of mesenchymal stem cells (MSCs) – show large variation between cells. In the course of towards effective in vitro cell propagation. differentiation process, expression profiles of individual cells become less variable. Also, we obtained Kersti Jääger, Angelika Fatkina, Anna Velts, Ester Orav significant upregulation of expression of components of and Toomas Neuman; Institute of Gene Technology, the chromatin modifying complex (e.g. SMARCA4, Tallinn University of Technology; Cellin Technologies, SMARCB1, SMARCC2), TAF complex (e.g. TAF7, LLC, Akadeemia tee 15A, Tallinn 12618, ESTONIA, TAF11, TAF15), general factors (e.g. [email protected] GTF2H1, GTF2E2, GTF2F2), coactivators and corepressors (e.g. NCOR2, NCOA3, NCOA6) in the Introduction process of differentiation of both AdMSCs and FBs. Multipotent mesenchymal stem cells (MSCs) have been Obtained results clearly demonstrate that AdMSCs and isolated from different adult tissues, including fat and FBs grown in vitro contain distinct subgroups with skin. Adipose tissue-derived MSCs (AdMSCs) possess different profiles. Presence of these stem cell-like properties such as self-renewal and subpopulations in the in vitro cultures during cell differentiation into variety of cell types including propagation and differentiation points to the need for adipocytes, osteoblasts and chondrocytes. Recent control of the subpopulations when used for therapeutic evidence suggests that similar differentiation potential is applications. possessed by tissue stromal cells (fibroblasts, FBs). However, molecular mechanisms of multipotency of AdMSCs and FBs have been shown to differentiate into AdMSCs and FBs are not well-characterized, especially adipocytes and osteoblasts in vitro. Little attention has the regulatory cascades at the early stages of development been paid on the molecular events related to the early into different phenotypes. The aims of the study were to stages of differentiation following induction. Our data (see Figure 1): show that key regulators of adipogenesis are induced 1) analyze gene expression patterns of proliferating differently in AdMSCs and FBs with a delayed induction AdMSCs and FBs at single-cell level using whole in FBs. For example, qRT-PCR analysis showed that transcriptome sequencing technology, PPARgamma mRNA induction has a delay of 3-7 days in 2) analyze regulatory mechanisms of the induction of FBs compared to AdMSCs. Analysis of osteogenic AdMSCs and FBs to differentiate into osteoblasts and differentiation did not reveal significant differences in the adipocytes with special focus on the dynamic expression profiles of key regulators of osteogenesis between of key regulators of differentiation, AdMSCs and FBs. However we observed significant 3) develop treatments to enhance propagation and differences in gene expression profiles between cells differentiation efficiency of AdMSCs and FBs in vitro. isolated from different patients. Studies focusing on early steps of differentiation will shed light on the key events controlling the switch of cells from undifferentiated state to committed cells and will contribute to development of effective techniques for adult stem cells manipulation in vitro.

Identification of signaling pathways and regulatory networks that control growth and differentiation of AdMSCs is essential to develop optimal in vitro expansion and differentiation conditions for these cells. Both, proliferation and differentiation of AdMSCs is

affected by the presence of different combinations of Figure 1. Overview of the project objectives growth factors in the culture media. We have developed serum-free culture conditions that stimulate proliferation Materials and Methods of AdMSCs compared to standard serum-containing AdMSCs and dermal FBs (isolated from the same media. Simultaneous activation of different signaling patients) were grown in vitro using variety of cell culture pathways has a significant effect on proliferation rate of conditions. Different molecular techniques including AdMSCs with no alteration of differentiation potential. qRT-PCR, Western blot and single-cell mRNA-seq technology were used to analyze molecular mechanisms Conclusions of growth and differentiation of cells. In vitro cultures of adult stem cells exhibit high degree of heterogeneity in the gene expression of individual cells as Results and Discussion well as in the proliferation and differentiation dynamics. We used single-cell mRNA-seq analysis to characterize However, via manipulation of different cellular signaling gene expression at different time points of osteogenic systems, cell populations with specific characteristics can differentiation of AdMSCs and FBs in vitro. Gene be developed for cell therapy applications.