Donor-dependent variation of human umbilical cord blood mesenchymal stem cells in response to hypoxic preconditioning and amelioration of limb ischemia

Figures

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  Fig. 1. Under hypoxic conditions, hUCB-MSC maintain stem cell characteristics and increase angiogenesis activity. a Immunostaining of Hif-1α was performed in hUCB-MSCs in normoxia and hypoxia. Graph indicates the quantification of Hif-1α expression. b Cumulative population doubling levels were measured to indicate the proliferation of hUCB-MSC under normoxic and hypoxic conditions. c Using flow cytometry, phenotype determination of hUCB-MSC under normoxia and hypoxia were analyzed. d Representative images of hUCB-MSCs differentiated into osteogenic, adipogenic, and chondrogenic lineages under normoxic and hypoxic conditions. Osteogenic differentiation is determined by optical density at 405 nm and Alizarin Red S, and adipogenic differentiation was determined by optical density at 500 nm and Oil Red O. e Effect of hUCB-MSCs cultured under normoxia and hypoxia on HUVEC migration in a co-cultured transwell system. Migrated HUVECs were stained with hematoxylin and DAPI. Graphs indicate counted migrated HUVECs. f Capillary-like tube formation assay of HUVECs co-cultured with hUCB-MSCs cultured under normoxic and hypoxic conditions. Tube formation assay analysis was carried counting total…

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  Fig. 2. Therapeutic effect of normoxic- and hypoxic-preconditioned hUCB-MSCs in a hindlimb ischemia animal model. Hindlimb ischemia was induced by ligations on the femoral artery and collateral vein. On Day 0, the hUCB-MSCs derived from different donors were injected after normoxic, short term and long term of hypoxic conditioning. a Scheme of HLI induction and cell treatment. b The HLI-induced mice were monitored on Days 1, 3, 7, 14, and 28, and photographic images of ischemic lesions were collected for gross examination. Functional recovery of cell-injected HLI mice was quantified by the (c) Tarlov and (d) Ischemia score systems. The results separately show the data from cell-injected groups for better legibility. In vivo experiments conducted under the same experimental conditions and whole-data of photographic images and functional quantifications, including negative and positive control groups are shown in Supplementary Figure S2. Five mice/group were used. *P < 0.05. Results are shown as the mean ± SD

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  Fig. 3. Histopathological assessment of ischemic lesions after normoxic and hypoxic-preconditioned hUCB-MSCs administration. On Day 28, mice were killed for histopathological evaluation. a Paraffin-embedded sections of muscle samples from HLI mice were stained with hematoxylin and eosin, and b muscle degeneration and c the number of infiltrated neutrophils were measured, bar = 200 μm. d The sections were stained with picrosirius red, and e muscle necrosis were measured, Scale bar = 200 μm. Four to five mice/group were used. ***P < 0.001. Results are shown as the mean ± SD

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  Fig. 4. Promoted hindlimb angiogenesis of the ischemic lesions show different effects among culture conditions and donors. a Representative image of CD31-positive blood vessels (green) from the adductor muscle section among groups. Scale bar = 50 μm. b Quantification of CD31-positive capillaries among groups. ***P < 0.001. Results are shown as the mean ± SD

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  Fig. 5. In vitro angiogenesis capacity is altered between culture condition and donor. a Capillary-like tube formation assay of HUVECs co-cultured with hUCB-MSC #55 or #64. Tube formation was quantified by total tube formation length and branch junction. Original version of images are shown in Supplementary Figure S3. b Migration assay using HUVECs co-cultured with hUCB-MSCs. Graphs show migrated HUVECs by total cell counting. c–e VEGF, PGE2, and PGI2 concentrations were measured from the cultured media of hUCB-MSCs by ELISA. Scale bars = 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001. Results are shown as the mean ± SD

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  Fig. 6. Genome-wide NGS analysis provides a clue to donor-dependent variations in stem cell therapy for limb ischemia. a–h Expression levels of transcriptomes in six samples N#55, SH#55, LH#55, N#64, SH#64, and LH#64 were analyzed by NGS analysis. The mRNA sequencing analysis was indicated normalized gene expression changes of angiogenic factors (VEGFA, VEGFB, VEGFC, ANG1, ANG2, ANG3 and ANGPTL4; color) and their receptors (VEGFR1, VEGFR2, VEGFR3, TIE1 and TIE2; gray), as well as additional markers (SLC2A3, ADM, and CDON) in both hUCB-MSC lines #64 and #55 as compared to the hypoxic-preconditioned SH- or LH#64 and SH- or LH#55, respectively

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  Fig. 7. Validation of selected genes expression by qPCR in low- and high-angiogenesis capacity hUCB-MSCs. a Validation of gene expression of genes selected form NGS (ANGPTL4, ADM, CDON, and SLC2A3) was analyzed by qPCR and show significant different expression pattern between low-angiogenesis capacity hUCB-MSCs and high-angiogenesis capacity hUCB-MSCs. b Capillary-like tube formation assay of HUVECs co-cultured with hUCB-MSCs #55, #64, #87, #132, #197, #202, #209 was performed and revealed two type of groups, low-angiogenesis capacity (#55, #87, #132, #202) and high-angiogenesis capacity (#64, #197, #209). The angiogenesis capacity of hUCB-MSCs was determined by quantification of tube formation length and branch junction. Scale bars = 100 μm. *P < 0.05, ***P < 0.001. Results are shown as the mean ± SD