Journal: bioRxiv

Article Title: Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

doi: 10.1101/2020.09.10.292409

Figure Lengend Snippet: ( a ) Illustrative summary of adult keratinocyte (KC), fibroblast (FB) and endothelial colony-forming progenitor cell (ECFC) propagation for testing self-assembly in vitro and in vivo. KCs and FBs were isolated from split-thickness skin explants. ECFCs were isolated from umbilical cord blood (I. + II). All cell types were propagated as monolayers under animal-serum free conditions generating app. 1 × 10 8 cells per cell factory (III.), before generating single-cell suspensions in human platelet lysate (hPL)-supplemented media promoting cell self-assembly into human skin organoids and vascularized human skin (IV.). ( b ) Morphology and cytokeratin 14 expression of four randomly selected KC preparations illustrating donor variability (Scale bar 400 µm). For cell transplantation, >99% pure cytokeratin 14 + keratinocytes were used.

Article Snippet: Thereafter, the cells were exposed to EGM-2/10% hPL with 260 ng/ml vascular endothelial growth factor (293-VE, R&D Systems) and 2 μM forskolin (F6886, Sigma) for 5 days prior to cell sorting.

Techniques: In Vitro, In Vivo, Isolation, Expressing, Transplantation Assay

Journal: bioRxiv

Article Title: Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

doi: 10.1101/2020.09.10.292409

Figure Lengend Snippet: ( a ) Immune-fluorescence showing CD90 expression of culture-expanded adult skin fibroblasts (FBs, blue), intracellular K14 expression of keratinocytes (KCs, green) and CD31 surface expression of endothelial colony-forming progenitor cells (ECFCs, red; all pseudo-colored). ( b ) Flow cytometry confirmed purity of isolated cells. ( c ) CFU assays showed donor-dependent clonogenicity of FBs and ECFCs (n = 3; mean ± SD). ( d ) KC colony on a feeder layer. ( e ) Vascular network formation after 12 hours on matrigel confirmed angiogenic potential of ECFCs (color-coded for automatic counting). ( f ) Primary skin FBs and KCs, but not ECFCs formed compact monotypic 3D spheroids (FBs = blue-, ECFCs= red-, KCs= green-labeled with nanoparticles). ( a, b, d-f ) Data from one of three independent donors shown. Scale bar = 100 µm.

Article Snippet: Thereafter, the cells were exposed to EGM-2/10% hPL with 260 ng/ml vascular endothelial growth factor (293-VE, R&D Systems) and 2 μM forskolin (F6886, Sigma) for 5 days prior to cell sorting.

Techniques: Fluorescence, Expressing, Flow Cytometry, Isolation, Labeling

Journal: bioRxiv

Article Title: Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

doi: 10.1101/2020.09.10.292409

Figure Lengend Snippet: ( a ) Triple cell type organization of fibroblasts, keratinocytes and endothelial cells as evaluated in endothelial growth medium (EGM) and serum-free keratinocyte growth medium (KGM). Light microscopy showed viable (green fluorescence viability dye) organoid formation promoted by platelet-derived growth factors (+hPL), whereas basal media without hPL or with basic supplements (+suppl.) did not support organoid formation. ( b ) Significantly more organoids were formed in the presence of hPL. One-Way-ANOVA and multiple comparison of three areas of two biological and three technical replicates; p values as indicated. ( c, d ) Live cell tracking in the 3D organization process with fluorescent nanoparticles (fibroblasts, blue; endothelia; red; keratinocytes, green). Organoid 3D assembly starting from initial stromal-vascular aggregation and followed by superficial anchorage of adult KCs, indicating well-organized organoids at app. 48 h after cell seeding, repeated in triplicates. Scale bar: 100 µm. ( e ) Proteome profiling of un-starved single FBs, ECFCs, KCs and 4-day assembled organoids after 12 hours stimulation in the absence (HCL control) or presence of IL17A. Z-scores (selected representative analysis).

Article Snippet: Thereafter, the cells were exposed to EGM-2/10% hPL with 260 ng/ml vascular endothelial growth factor (293-VE, R&D Systems) and 2 μM forskolin (F6886, Sigma) for 5 days prior to cell sorting.

Techniques: Light Microscopy, Fluorescence, Derivative Assay, Comparison, Cell Tracking Assay, Control

Journal: bioRxiv

Article Title: Self-assembly of progenitor cells under the aegis of platelet factors facilitates human skin organoid formation and vascularized wound healing

doi: 10.1101/2020.09.10.292409

Figure Lengend Snippet: Single-cell suspensions (adult keratinocytes, fibroblasts and endothelial cells) re-suspended in 10% hPL- or 10% FBS-supplemented media were transplanted and grafts collected days 14 or 28 . ( a-t ) Histology of transplants in the absence or presence of ECFCs, supported by hPL or FBS, day 14 post grafting; compared to ( a,e,i,n,r ) healthy human control skin. ( a-d ) Anti-human vimentin (hVIM) confirmed the human origin of the dermis and stratified skin organization; DAPI + nuclei, white. ( e-h ) Anti-Ki67-labelled proliferating cells (brown). ( i-m ) Polarized light-activated collagen fibers exclusively in control skin and hPL-supported, not in FBS-driven transplants (n-q) . Masson-Goldner trichrome (MG3C) histochemistry showed vessel enrichment when ECFCs were co-transplanted with fibroblasts/keratinocytes in the presence of hPL (+ECFCs/hPL) compared to transplants without ECFCs (-ECFCs/hPL) showing occasional murine vessel sprouting. Murine erythrocytes (red) confirmed blood circulation inside vessels. (r-t) Anti-human CD31 verified human vessel origin in +ECFCs/hPL. ( n-t ) Dotted red arrows = murine vessels. Filled arrowheads = human vessels. ( a-q ) Scale bar 100 µm. One out of three independent grafts per group shown. ( u ) Quantification showing significantly increased epidermal thickness in –ECFCs/FBS compared to –ECFCs/hPL. ( v ) Dermal quality score was significantly increased in hPL-compared to FBS-supported transplants and by ECFC presence day 14 after transplantation. ( w ) Vessel number in grafted cell-derived self-organized human dermis was significantly increased day 14 after ECFC co-transplantation. ( u-w ) One-Way-ANOVA, multiple comparison of three biological and three technical replicates; p values as indicated.

Article Snippet: Thereafter, the cells were exposed to EGM-2/10% hPL with 260 ng/ml vascular endothelial growth factor (293-VE, R&D Systems) and 2 μM forskolin (F6886, Sigma) for 5 days prior to cell sorting.

Techniques: Control, Transplantation Assay, Derivative Assay, Comparison

Journal: PLoS ONE

Article Title: Efficient Commitment to Functional CD34+ Progenitor Cells from Human Bone Marrow Mesenchymal Stem-Cell-Derived Induced Pluripotent Stem Cells

doi: 10.1371/journal.pone.0034321

Figure Lengend Snippet: (A-I) Hematopoietic CFU formation and hematopoietic lineages assay for the potential of CD34+ progenitor cells. (A) Measurement for CFU potential of CD34+ cells derived from hBMMSC-iPSCs compared to that from hFib-iPSCs. (B-F) Various types of CFUs were observed with a reversed telescope as BFU-E (B), CFU-E (C), CFU-GM (D), CFU-GEMM (E) and CFU-G (F) when CD34+ progenitor cells derived from hBMMSC-iPSCs were seeded into CFU forming culture system to assess the short-term differentiation capabilities of hematopoietic progenitors. (G-I) Morphologies of the hematopoietic lineages derived from CFU culture of CD34+ progenitor cell commitment to hematopoietic cells observed with a reversed telescope (Olympus), including erythroid from BFU-E (G), granulocyte from CFU-G (H) and macrophage, granulocyte, erythroid, megakaryocyte from CFU-GEMM (I). (J-L) Analysis of endothelial cell potential of CD34+ progenitor cells derived from hBMMSC-iPSCs. Some of CD34+ progenitor cells cultured with EGM-2 were positive to CD31 (J) and VE-CADHERIN (K). When treated with VEGF-A on Matrigel for 3 days, vascular-like structures were photographed (L).

Article Snippet: For the assay of endothelial cell potential, CD34+ progenitor cells were plated onto fibronectin-coated (Invitrogen) plates in endothelial cell growth medium EGM-2 (BD Falcon).

Techniques: Derivative Assay, Cell Culture