Induced Mesenchymal Stromal Cells (iMSCs) Derived via Neural Crest and Cytotrophoblast Lineages Showed Distinct Transcriptomic Profile and Extracellular Vesicle Production

Introduction: Multipotent mesenchymal stromal cells (MSCs) have shown promises for tissue repair and regeneration, autoimmune diseases, and chronic disorders. Primary MSCs (pMSCs) collected from different tissue sources show similar cell morphology, marker identity, and multi-lineage differentiation capacity, but exhibit variations in growth rate, transcriptomic profile, secretome signature, anti-inflammatory and immunomodulatory capacities. Such variability might originate from donor genetic and epigenetic background, tissue preparation techniques, cell culture and priming conditions, thus highlighting the need for standardization in both fundamental MSC biology and translational MSC therapy. The differentiation of MSCs from pluripotent sources (iMSCs) offers opportunities for scalable biomanufacturing of these cells. Recent progress suggests that controlled lineage specification of iMSCs through defined stepwise differentiation processes gave rise to end-stage iMSC subtypes with developmental lineage specificity. We hypothesize that pMSCs’ tissue specificity can be traced back to the lineage specification during embryonic development that will be epigenetically inherited by the MSCs. Hence, we will mirror the developmental blueprint of embryos to differentiate iMSCs through two different intermediate cell stages of neural crest (NC-iMSCs) and cytotrophoblast (CT-iMSCs) using serum-free chemical-defined media. We also performed a systematic comparison of iMSCs from different lineages and pMSCs from different tissues to define the developmental signatures of iMSCs and understand their commonalities and differences with pMSCs. This study demonstrated that different iMSC subtypes and priming conditions affected EV production, exosome protein expression, and cytokine cargo, highlighting the importance of generating lineage specific iMSCs to improve their therapeutic potential.

Materials and

Methods: We characterized our NC-iMSCs and CT-iMSCs using immunostaining against typical MSC markers of CD73, CD105, CD90, and CD44 and tri-lineage differentiation of adipogenesis, osteogenesis, and chondrogenesis. We performed bulk RNA sequencing (RNAseq) to understand the similarity and difference between lineage-specific iMSC subtypes and source-specific pMSC subtypes, including bone marrow pMSCs (BM-pMSCs), adipose tissue pMSCs (AD-pMSCs), dental pulp pMSCs (DP-pMSCs), umbilical cord pMSCs (UC-pMSCs), chorionic villi pMSCs (CV-pMSCs), and chorionic plate pMSCs (CP-pMSCs). To better determine the iMSC development and heterogeneity, we performed single-cell RNA sequencing (scRNAseq) and pseudo-time trajectory analysis (Monocle 3) on NC-iMSCs and CT-iMSCs. With a particular interest in extracellular vesicle (EV) biomanufacturing from iMSCs, we primed both NC-iMSCs and CT-iMSCs using either Lipopolysaccharide (LPS) or Cell Stimulation Cocktail (CSC). The iMSC-EVs were characterized by nanoparticle tracking analysis (NTA), western blot on exosome markers, and Luminex assay on inflammation-related protein cargos.

Results, Conclusions, and Discussions: We confirmed successful iMSC differentiation from both neural crest and cytotrophoblast lineages based on positive immunostaining of typical MSC markers and negative of pluripotent markers. Our iMSCs exhibited differentiation potentials into adipogenic (oil red O), osteogenic (osteocalcin) and chondrogenic (aggrecan) lineages. Using flow cytometry, we found that NC-iMSCs had a higher yield in ~90% of CD73+/CD90+/CD105+ cells than the CT-iMSCs with ~85% of CD73+/CD90+ cells, while ~75% of CD105+ cells. From scRNAseq results, we observed that NC-iMSCs and CT-iMSCs overlapped with each other. Entire cell populations were divided into 5 clusters: cluster 1-4 highly expressed all MSC surface markers and transcription factors, while cells from cluster 5 were non-iMSCs (only ~5%). Cycling pre-MSCs (cluster 1) were highly enriched in genes associated with cell division and DNA replication, while MSCs (cluster 2) and osteochondro-progenitors (cluster 3) were highly enriched in genes associated with cell differentiation, tissue development, and cell-ECM interaction. Stimulated MSCs (cluster 4) were enriched in genes associated with cell stress and cell senescence. Next, we compared two lineage-specific iMSCs and six tissue-specific pMSCs. Key genes associated with early embryonic and ectodermal development were upregulated in the iMSCs, while the genes upregulated in the pMSCs were more associated with cell-ECM interactions. In addition, NC-iMSCs upregulated the genes associated with musculoskeletal development, while CT-iMSCs upregulated the genes associated with tumor suppression and immunomodulation. Last, we collected and purified the small EVs (sEVs, < 200 nm) from cell culture media. We observed a higher sEV concentration from CT-iMSCs primed with CSC than the NC-iMSCs. The sEVs from both CT-iMSCs and NC-iMSCs had robust comparable expression of CD63 and CD81, while sEV from CT-iMSCs had higher expression of CD9 than the ones from NC-iMSCs. More surprisingly, the sEVs from the CT-iMSCs highly expressed HSP90α/β protein, which was not found from the sEVs produced by the NC-iMSCs.

We revealed that MSCs were enriched in genes related to osteogenesis, immunomodulation, and cell-ECM interaction. NC-iMSCs had a higher MSC purity and stronger osteogenic potential than CT-iMSCs. However, CT-iMSCs had better EV production and immunomodulatory function than NC-iMSCs, making CT-iMSCs a better candidate for therapeutic EV biomanufacturing.

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