SEARCH

SEARCH BY CITATION

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
stem1560-sup-0001-suppfig1.tif27407KSupplementary figure 1: Suffering state of freshly isolated mouse CM (A) Annexin V and 7AAD staining of mouse CM just after heart isolation and prior coculture indicating that around 80 % of them are necrotic and 15% enter in apoptosis. (B) CM staining with CellRox dye showing that most of freshly isolated CM are in an oxidative stress state. (A-B) Experiments were repeated at least n=5 fold.
stem1560-sup-0002-suppfig2.tif32313KSupplementary figure 2: Secretome changes of hMADS according to the seeding density. Relative transcriptional gene expression changes (qPCR) of hMADS after 24 hours coculture with CM (1HM+1CM:1HM) compared to that of hMADS seeded in double number (2HM:1HM). Results are expressed in relative means ± SD of 3 independent experiments. *p<0.05, **p<0.01 and ***p<0.001.
stem1560-sup-0003-suppfig3.tif30521KSupplementary figure 3: Latrunculin A or nocodazole do not affect phagocytic and endocytic activities of hMADS. (A) Flow cytometry analysis of calcein and mitotracker transfers from CM to hMADS prior (CCT0) and after 24 hours of coculture without (CC) or in presence of 0.4μm (TW 0.4μm) or 1μm (TW 1μm) pore size transwell insert. Red and black dots represent CM and hMADS cell populations, respectively. (B) Microscopic immunofluorescence (upper panel) and flow cytometry analysis (lower panel) showing phagocytosis of Ph rodo Red S.aureus bioparticles by hMADS cells, treated or not during 24 hours with either latrunculin A or nocodazole. (C) Microscopic immunofluorescence (upper panel) and flow cytometry analysis (lower panel) showing endocytosis of pHrodo green dextran by hMADS cells, treated or not during 24 hours with either latrunculin A or nocodazole. (B-C) As expected, phagocytosis and endocytosis are inhibited when cells are exposed at 4°C. Experiments were independently repeated n=4 fold.
stem1560-sup-0004-suppfig4.tif27740KSupplementary figure 4: HMADS and CM do not communicate through functional gap junctions (A) Flow cytometry analysis of calcein transfer from CM to hMADS in 24hour-coculture. CM are loaded with calcein prior coculture. Calcein transfer occurs from CM to stem cells (middle dot blot) and is insensitive to presence of 18α-GA, a gap junction blocker (right dot blot). Histogram overlays detecting presence of calcein into hMADS (black and gray lines correspond to 18α-GA treated- and control cocultured hMADS cells, respectively. Filled gray histogram corresponds to naïve hMADS cells) (B) Flow cytometry analysis indicating that calcein is not transferred from hMADS to CM in 24hour-cocultures. hMADS are loaded with calcein prior coculture. Histogram overlays confirm lack of calcein into 24 hour-cocultivated CM (black and gray lines correspond to 18α-GA treated and control cocultivated CM, respectively. Filled gray histogram corresponds to CM alone. (A-B) Experiments were independently repeated n=4 fold.
stem1560-sup-0005-suppfig5.tif39922KSupplementary figure 5: Inflammation affects TNT-mediated effects while human engrafted cocultured and naive hMADS cells are similarly cleared by immunocompetent mice. (A) Average number of heterologous TNT connections in 24 hour cocultures in which hMADS cells (HM) were previously primed for 18 hours with different concentrations (10, 20 and 50 ng/ml) of TNF α or IFN ?. Data represent the mean ± SD of 4 independent experiments. *p<0.05, ** p<0.01, ***p<0.001. (B) Relative variation of transcriptional gene expression (qPCR) after 24 hours of coculture containing hMADS cells previously primed with 50 ng/ml of TNF α or 20 ng/ml IFN ? (black rectangle) by reference to untreated cocultures (white rectangle). Data represent the mean ± SD of 4 independent experiments. *p<0.05, ** p<0.01, ***p<0.001. (C) Left panel: heart immunohistochemistry at day-3 post-infarction showing hLamin A/C+ cells. Nuclei were counterstained with DAPI. Scale bar, 50μm. Right panel: histogram representing the number of hLamin A/C+ cells per heart after treatment with hMADS cells alone (HH) or in coculture (CC) at day3 post infarction. Data represent the mean ± SD of n=5 animals per group. “ns” means no significant.
stem1560-sup-0006-suppfig6.tif19431KSupplementary figure 6: Improved myocardial regeneration of co-cultivated hMADS is not associated with transdifferentiation or permanent cell fusion. (A) Heart immunohistochemistry at day-3 post-infarction showing that hLamin A/C+ cells were CD31-, GATA-4- and cTnT-. Scale bar, 50μm. (B) FISH with mouse and human Cot-1 DNA probes showing the lack of fused cells in mice hearts injected with hMADS grown alone or in co-culture at day 3 post-infarction. Scale bar, 20μm. (A-B) Nuclei were counterstained with DAPI.
stem1560-sup-0007-suppfig7.tif28334KSupplementary figure 7: CM having higher amount of human stem cell mitochondria are less susceptible to apoptosis. (A) Flow cytometry analysis of 24 hour-coculture in which hMADS cells were pre-loaded with WGA (an undiffusible dye) and mitotracker green. Upper panel: left dot blot, unstained CM; middle dot blot, hMADS preloaded with WGA and mitotracker green, right dot blot, mitochondrial transfer in 24 hour cocultures indicating the existence of two CM populations differing with regards to their abundance in stem cell mitochondria, the P1 population expressing lesser amount of human stem cell mitochondria than the P2 one. Lower panel: flow cytometry analysis showing that (right dot-blot) that CM cultured alone during 24 hours were all apoptotic, (middle dot blot) hMADS cultured alone were not apoptotic and (right dot blot) that P2 population of CM contained higher level of annexin V negative cells than P1 one. (B) Upper panel: flow cytometry showing that CM are devoid of human stem cell mitochondria in 24 hour-cocultures performed with (left dot blot) 0.4μm or (right dot blot) 1μm transwell pore size. Lower panel: flow cytometry analysis showing that CM in 24 hour- cocultures performed with (left dot blot) 0.4μm or (right dot blot) 1μm transwell pore size were all apoptotic. (C) Histogram overlays showing that CM in normal 24 hour-cocultures express decreased levels of annexin V (black histogram) compared to CM cultured alone (filled gray histogram) or in indirect cocultures performed with either 0.4μm (gray histogram) or 1μm (dotted gray histogram) pore size transwell. (A-C) Experiments were independently repeated n=4 fold.
stem1560-sup-0008-suppinfo.docx33KSupporting Information
stem1560-sup-0009-supptab1.docx13KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.