In vitro generation of vascular wall-typical mesenchymal stem cells

Stem cell-based therapy is a promising option for the treatment of various diseases. Herein, mesenchymal stem cells (MSCs), which are adult multipotent stem cells, are of particular interest. Once applied, MSCs could migrate to the site of tissue damage replacing lost or damaged cells by differentiation; but more importantly MSCs are able to secrete numerous different growth factors and cytokines (paracrine mechanism) mediating tissue regeneration and even protecting healthy tissue from (further) destruction. However, tissue-resident stem cells are rather low in numbers, and isolated MSCs need rigorous in vitro expansion. Therefore, more easily accessible sources are needed. An alternative method demonstrates the invitro differentiation of MSCs from induced pluripotent stem cells (iPSCs) or adult somatic cells such as fibroblasts. And in terms of manufacturing exogenous MSCs with superior repair capabilities, tissue-specific MSCs might be logically more efficient, when therapeutically applied to a certain injured tissue, than MSCs derived from a different origin, e.g. the bone marrow. Among the different MSC sources, MSCs residing within the wall of larger blood vessels, so-called vascular wall-resident MSCs (VW-MSCs), are predestined to address vascular damage, due to their tissue-specific action. In order to generate a huge amount of donor-independent VW-MSCs for therapeutic approaches in vitro, a proof of principle attempt was made by introducing a recently identified VW-MSC specific gene code (the “HOX-code”) into murine induced pluripotent stem cells (iPSCs), which finally yielded cultures of VW-typical MSCs (78, 147).

The established and optimized protocol for the in vitro generation of VW-MSCs from murine iPSCs, which was used as the basis for the following work investigated during the present thesis, was summarized within the first manuscript (Steens et al., Methods Mol Biol. 2020). Herein, dermal tail-tip fibroblasts from a Nestin-GFP transgenic mice, in which GFP was integrated into the Nestin locus to facilitate lineage tracing after MSC differentiation, were reprogrammed using the four Yamanaka factors (NEST-iPSCs). Following clonal expansion and detailed characterization of NEST-iPSCs, a lentiviral vector encoding a small set of VW-MSC specific HOX genes, namely HOXB7, HOXC6 and HOXC8, was then used to induce MSC differentiation. The generated murine VW-MSCs highly expressed Nestin-GFP and showed all the classical MSC characteristics in vitro such as plastic adherence, typical MSC marker expression and the potential to differentiate into mesodermal lineages as well as into vascular cells. Thus, a protocol for the in vitro generation of (murine) VW-MSCs by using VW-MSC specific transcription factors was successfully established (manuscript 1: Steens et al., Methods Mol Biol. 2020).

The central aim of the present thesis was to translate this approach to human cells. In terms of being a master regulator for VW-MSC identity, the triple combination of the VW-MSC specific HOX genes (the HOX-code comprising HOXB7, HOXC6 and HOXC8) was used to directly convert human donor cells into vascular wall-typical MSCs. In the second work therefore, primary human fibroblast cultures derived from healthy donors were transduced with a lentiviral supernatant introducing this VW-MSC specific HOX code. The transduced cells were purified by flow cytometry sorting and then expanded. Generated VW-MSCs showed an ectopic expression of the introduced HOX genes, classical MSC morphology, and MSC-typical marker expressions. Of note, an increased colony-forming capacity and differentiation potential turned out to be the most specific features that discriminate MSCs from fibroblasts, because classical features like MSC marker expression were not sufficient to distinguish both cell types based on their shared mesenchymal origin. Furthermore, fibroblast-derived VW-MSCs displayed the VW-MSC typical potential to differentiate into vascular mural cells (pericytes and smooth muscle cells). Comparative global gene expression and DNA methylation analysis finally confirmed that fibroblast-converted VW-MSCs adapted a VW-MSC phenotype. Concerning the stability of the generated phenotype, a doxycycline-inducible expression system of the HOX code was used revealing that the introduced HOX protein expressions as well as the acquired clonogenicity and differentiation potential of the generated VW-MSCs were retained (up to four weeks following doxycycline removal). The therapeutic potential of fibroblast-derived VW-MSCs was further analyzed in vitro and invivo. Invitro, the generated VW-MSCs suppressed lymphocyte proliferation, enhanced wound healing and mediated radioprotection especially for vascular elements. Similar to “real” VW-MSCs, generated VW-MSCs secreted various (growth) factors including angiogenic cytokines, which further highlights that (i) the trophic nature of MSCs mainly depends on the cellular origin, and (ii) that these cells are predestinated to address vascular damage. Finally, using a mouse model of radiation-induced pneumopathy, the generated VW-MSCs were able to reduce vascular damage in respective lungs, thereby limiting inflammation as well as fibrosis development after therapeutic application (manuscript 2: Steens et al., Cell Ml. Life Sci. 2020).

In a parallel approach, the protocol for the in vitro generation of VW-MSCs from murine iPSCs (manuscript 1) is transferred and optimized for human iPSCs (manuscript in preparation). Human iPSCs were therefore already successfully transduced (with vectors coding for the HOX-code, as well as an empty vector controls), purified by flow cytometry and expanded in culture (not shown). Unfortunately, due to technical challenges there is a severe delay in performing respective follow up experiments. Therefore, the translational approach for the in vitro generation of VW-MSCs using human iPSCs, and in particular the investigations about the therapeutic potential, could not be finalized within the period of the present thesis.

Taken together, gene-transfer of the VW-MSC specific HOX genes HOXB7, HOXC6 and HOXC8 successfully induced differentiation of murine iPSCs into VW-MSCs and even converted human fibroblasts into therapeutically-active VW-MSCs. Herein, the HOX-code seems to be a master-regulator of VW-MSC identity. Likewise, expression of this HOX-code (together with VW-MSC specific features) serves as an additional characteristic to discriminate VW-MSCs from phenotypical similar cells like fibroblasts or from vascular cells (78). Although the protocol for the production of VW-MSCs from human iPSCs needs to be refined in further investigations. However, both manuscripts of the present thesis already highlight that the invitro generation of VW-MSCs is a promising method to generate a huge amount of patient-specific MSCs for therapeutic applications.

Despite the fact that therapeutically applied MSCs, particularly VW-MSCs, present a valuable therapeutic option for tissue regeneration of diseased lungs, less is known about the true function of endogenous MSCs within lungs, and particularly of their niche. Therefore, in the third work the VW-MSC specific HOX code was further used to unravel whether VW-MSCs can be found in other tissues within the vascular niche, particularly within lungs (manuscript 3: Steens et al., Stem Cells Transl. Med., 2020). Immunohistochemical staining of human normal lung tissues demonstrated that single CD44(+) CD146(+) VW-MSCs can be found within the adventitial vasculogenic zone of adult lung blood vessels. Isolated primary lung-resident MSCs (LR-MSCs) further displayed a typical MSC morphology and typical MSC characteristics in vitro. Of note, the differentiation potential and the colony-forming capacity were highly similar to those of VW-MSCs. Most importantly, LR-MSCs highly expressed the VW-MSC specific HOX code strongly suggesting that LR-MSCs should be considered as VW-MSCs. These LR-MSCs could even be involved in lung diseases, e.g. lung cancer and could act here as tumor modulators. Accordingly, significant increases of VW-MSC numbers were estimated within lung cancer tissue and expressions of the (VW-) MSC marker CD44 as well as of the HOX genes correlated with a worse overall survival of respective patients (manuscript 3: Steens et al., Stem Cells Transl. Med., 2020).

Conclusively, whereas VW-MSCs within human lungs (LR-MSCs) usually contribute to a healthy lung homeostasis bearing the capacity to suppress inflammation and to promote lung repair, VW-/LR-MSC seem not to appear sufficient for tissue protection or repair upon lung damage or particularly lung cancer as investigated here. These findings provide implications for understanding the role of (VW-) MSC in normal lung physiology and pulmonary diseases, as well as for the rational design of additional therapeutic approaches.


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