{'fraction_non_alphanumeric': 0.046844404277752674, 'fraction_numerical': 0.014309383943364964, 'mean_word_length': 5.415458937198068, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Direct conversion of human fibroblasts into therapeutically active vascular wall-typical mesenchymal stem cells

Cell-based therapies using adult stem cells are promising options for the treatment of a number of diseases including autoimmune and cardiovascular disorders. Among these, vascular wall-derived mesenchymal stem cells (VW-MSCs) might be particularly well suited for the protection and curative treatment of vascular damage because of their tissue-specific action. Here we report a novel method for the direct conversion of human skin fibroblasts towards MSCs using a VW-MSC-specific gene code (HOXB7, HOXC6 and HOXC8) that directs cell fate conversion bypassing pluripotency. This direct programming approach using either a self-inactivating (SIN) lentiviral vector expressing the VW-MSC-specific HOX-code or a tetracycline-controlled Tet-On system for doxycycline-inducible gene expressions of HOXB7, HOXC6 and HOXC8 successfully mediated the generation of VW-typical MSCs with classical MSC characteristics in vitro and in vivo. The induced VW-MSCs (iVW-MSCs) fulfilled all criteria of MSCs as defined by the International Society for Cellular Therapy (ISCT). In terms of multipotency and clonogenicity, which are important specific properties to discriminate MSCs from fibroblasts, iVW-MSCs behaved like primary ex vivo isolated VW-MSCs and shared similar molecular and DNA methylation signatures. With respect to their therapeutic potential, these cells suppressed lymphocyte proliferation in vitro, and protected mice against vascular damage in a mouse model of radiation-induced pneumopathy in vivo, as well as ex vivo cultured human lung tissue. The feasibility to obtain patient-specific VW-MSCs from fibroblasts in large amounts by a direct conversion into induced VW-MSCs could potentially open avenues towards novel, MSC-based therapies. Electronic supplementary material The online version of this article (10.1007/s00018-019-03358-0) contains supplementary material, which is available to authorized users.


Supplemental Figure Legends
Supplementary Figure S1 Characterization of control-transduced fibroblasts and iVW-MSCs (A) Representative scatter plots from the HOX-and control-vector (Ctrl) transduced fibroblasts only expressing Turquoise2 are shown. The gated cell population (P5) was isolated by FACS. (B) Cell viability of cultured iVW-MSCs and control fibroblasts was assessed using a WST-1 reagent-based tetrazolium reduction assay. Proliferation was further analyzed with the crystal violet assay. Data are shown as means ± SEM of three independent experiments measured in quadruplets each. (C) Single cells suspension of control-transduced fibroblasts (CtrlTdFib) and iVW-MSCs were analyzed by FACS with indicated MSC marker antibodies (biological replicates: n=6-8 for each epitope and group). (D) Differential MSC marker gene expression levels in iVW-MSCs and control-transduced fibroblasts (CtrlTdFib) as compared to ex vivo isolated hITA (human internal thoracic artery)-derived VW-MSCs (positive control) and primary (non-transduced) fibroblasts (donor cells; negative control) were further confirmed by qRT-PCR. Relative amounts of transcripts of the indicated genes were determined by qRT-PCR (biological replicates: n=4-6 per group and gene; P by two-way ANOVA, followed by post-hoc Tukey's multiple comparisons test: *P ≤ 0.05; **P ≤ 0.01).

Supplementary Figure S2
Trilineage differentiation potential The ability to differentiate into mesodermal linage cells was investigated for primary fibroblasts (FIB), control-transduced fibroblasts (CtrlTdFib), iVW-MSCs and hITA-derived VW-MSCs. Differentiation was observed within 14 days after induction of differentiation (DM) as shown by Oil red staining (adipocytes), by histochemical staining for alkaline phosphatase (ALP) of osteocytes, or by immunocytochemistry for collagen type II expression in chondrocytes. The trilineage differentiation potential was confirmed for iVW-MSCs and for hITA-derived VW-MSCs (positive control), while primary fibroblast as well as control-transduced fibroblast lack that differentiation potential.   Supplementary Figure S5 Therapy with iVW-MSCs improved leukocytes blood levels of WTI mice Total white blood cell counts as well as differential blood cell parameters (lymphocytes, monocytes, and granulocytes) were analyzed using peripheral blood samples (EDTA blood) within the inflammatory phase of WTI mice at 3 weeks (A; pneumonitic/acute phase) and 25 weeks (B; fibrotic/ chronic phase) after irradiation with or without therapeutically applied iVW-MSCs and control-transduced fibroblasts (CtrlTdFib) (n= 10-15 mice per group). C Peripheral blood samples were obtained 25 weeks after irradiation and circulating cyan-positive cells were quantified via flow cytometry. Data are presented as mean ± SEM (Ctrl: n=10 mice; HOX: n=17 mice). Data are presented as means ± SEM; *P ≤ 0.05.

Supplementary Figure S6
Potential VW-MSCs-derived growth factors and cytokines accounting for a paracrine action (A) Transcriptome heatmaps of growth factor and cytokine encoding genes extracted from the global gene expression profiles. Relative transcript amounts of exosome-associated surface markers and indicated immunomodulating genes are shown as boxplots (biological replicates: n=4 per group). (B) Differential candidate gene expression levels of known VW-MSC factors involved in tissue regeneration [1][2][3] in iVW-MSCs and control-transduced fibroblasts (CtrlTdFib) as compared to ex vivo isolated hITA (human internal thoracic artery)-derived VW-MSCs (positive control) and primary (non-transduced) fibroblasts (donor cells; negative control) were further confirmed by qRT-PCR. Relative amounts of transcripts of the indicated genes were determined by qRT-PCR (biological replicates: n=6-8 per group and gene; P by two-way ANOVA, followed by post-hoc Tukey's multiple comparisons test: *P ≤ 0.05).
Supplementary Figure S7 VW-MSCs-specific paracrine action affects the cell cycle of cultured endothelial cells Cell cycle phases of endothelial cells treated with and without irradiation and subsequent conditioned medium (CM) treatment was analyzed 24 hours after treatment. Data are shown as means ±SEM of three independent experiments measured in duplicates each. ****, #### P ≤ 0.0001, ***, ### P ≤ 0.001, **, ## P ≤ 0.01 *, # P ≤ 0.05, by two-way ANOVA followed by post-hoc Tukey's test. Significances (each P ≤ 0.0001) of the radiation-induced G1 reductions and the G/M arrests for each treatment condition were not indicated.

Supplementary Tables
Supplementary Table 1 Oligonucleotides used for qRT-PCR