Journal: Microsystems & Nanoengineering

Article Title: Modular operation of microfluidic chips for highly parallelized cell culture and liquid dosing via a fluidic circuit board

doi: 10.1038/s41378-020-00216-z

Figure Lengend Snippet: a Live-cell fluorescence images (one image per four chambers) showing an overview of all the chambers after 3 days of culturing GFP-expressing HUVECs. b Live-cell fluorescence image of GFP-expressing HUVECs in chambers 6 and 7. c Fluorescence image of fixed HUVECs with the cell F-actin and nuclei stained with ActinRed and NucBlue, respectively. d HUVECs after seeding and subsequent monolayer formation. The cells were seeded at a high cell density (i) and confluent on day 1 (ii). The monolayer was still intact on day 3 (iii) but began to deteriorate on day 4 (iv). At this point, the medium in the supply vial was replaced. The cells showed signs of recovery on day 5 (v) as the monolayer started to reform. The red dots in (iv) and (v) mark the cells counted in the region of interest. e Cells in ten chambers were counted in regions where the monolayer had deteriorated on day 4 (98 h) and after 16 h of recovery (114 h). The black dots represent the mean cell count, and the error bars represent the standard deviation. The cell number increased by an average of 33% in these areas

Article Snippet: Live-cell images of the GFP-expressing HUVECs (Angio-Proteomie, USA) were taken with an EVOS FL cell imaging system using the GFP filter cube.

Techniques: Fluorescence, Expressing, Staining, Cell Counting, Standard Deviation

Journal:

Article Title: Major Histocompatibility Complex-I Expression on Embryonic Stem Cell-Derived Vascular Progenitor Cells Is Critical for Syngeneic Transplant Survival

doi: 10.1002/stem.475

Figure Lengend Snippet: Embryonic stem cell (ESC) lineage differentiation and the effect of IFNγ on MHC-I expression. (A): ESCs were cultured in the presence of BMP-4 for 3.25 days. After this time, we detected a discrete population of Bry+Flk-1+ (Aa) cells that was also observed to be predominantly MHC-I negative (Ab). On return to culture and in the presence of VEGF for further 7 days, a population of VE-CAD+ endothelial progenitors (Ac) was identified, with extremely few of these cells also coexpressing MHC-I (0.18% ± 0.09%, n = 6) (Ac). When IFNγ was included in the culture medium for 2.5 days before final harvest, the majority of VE-CAD+ cells became MHC-I positive (87.3% ± 5.24%, p < .0001 vs. no IFNγ) (Ad). Alternatively, ESCs could be directly differentiated into Bry+Flk-1+MHC-I+ cells by the initial presence of BMP-4 and IFNγ (Ae and Af), although, a significant number of Bry+Flk-1+ cells remained MHC-I negative (Ag). Despite prior IFNγ treatment, Bry+Flk-1+ cells that were returned to culture in the presence of VEGF were able to continue differentiation into VE-CAD+ cells (Ah), although MHC-I expression was lost in this instance. (B): Immunohistochemistry staining of VE-CAD+ endothelial progenitors derived from Bry+Flk-1+ cells (as identified in panel Ac) showing positive staining for both VE-CAD and CD31. Scale bar = 10 μm. (C): To ensure that IFNγ did not perturb surface antigen expression, immunohistochemistry was performed on the VE-CAD+MHC-I− cell population indicated in (Ah) for CD31 and VE-CAD, revealing an identical staining pattern to IFNγ-naïve VE-CAD+MHC-I− cells (B). Scale bars = 10 γm. Abbreviations: BMP-4, bone morphogenetic protein-4; Bry-GFP, brachyury-green fluorescent protein; IFN-γ, interferon-γ; MHC-I, major histocompatibility complex; VE-CAD, VE-cadherin; VEGF, Vascular endothelial growth factor.

Article Snippet: Cells were isolated, washed, and then incubated with the following primary antibodies for 60 minutes: Flk-1 (#14-5821, eBioscience, San Diego, CA, http://www.ebio-science.com/ ), VE-CAD (#555289, BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/index_us.shtml ), PE-conjugated anti-MHC-II antibody (#130-091-368, Miltenyi Biotec, Auburn CA), PE-conjugated rat anti-mouse inter-cellular adhesion molecule 1 (ICAM) (ab24869, abcam), PE-conjugated rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1) (ab24853, abcam), and Biotin-conjugated H-2Db (#553572, BD Pharmingen).

Techniques: Expressing, Cell Culture, Immunohistochemistry, Staining, Derivative Assay

Journal:

Article Title: Major Histocompatibility Complex-I Expression on Embryonic Stem Cell-Derived Vascular Progenitor Cells Is Critical for Syngeneic Transplant Survival

doi: 10.1002/stem.475

Figure Lengend Snippet: Matrigel plug neovascularization assay and embryonic stem cell (ESC)-derived cell integration into new vessels. Fluorescence stereo-microscopic imaging revealed that while VE-CAD+MHC-I− cells in Matrigel exhibited new vessel formation (A), significantly more new vessel formation was observed for VE-CAD+MHC-I+ cells in Matrigel (B). Vessels were stained with FITC-dextran for 10 minutes before harvesting and blood flow was analyzed by quantitating FITC-dextran. VE-CAD+MHC-I+ plugs were found to contain significantly more FITC-dextran than VE-CAD+MHC-I− plugs (***, p < .001, VE-CAD+MHC-I+ vs. VE-CAD+MHC-I−, both n = 5) (C). Immunostaining revealed both DsRed/CD31 (D) and DsRed/α-smooth muscle actin double positive cells (E) in vessel structures, suggesting that ESC-derived VE-CAD+MHC-I+ cells integrated into the new vasculature in the form of endothelial and smooth muscle cells. Abbreviations: FITC, fluorescein isothiocyanate; MHC-I, major histocompatibility complex; VE-CAD, VE-cadherin.

Article Snippet: Cells were isolated, washed, and then incubated with the following primary antibodies for 60 minutes: Flk-1 (#14-5821, eBioscience, San Diego, CA, http://www.ebio-science.com/ ), VE-CAD (#555289, BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/index_us.shtml ), PE-conjugated anti-MHC-II antibody (#130-091-368, Miltenyi Biotec, Auburn CA), PE-conjugated rat anti-mouse inter-cellular adhesion molecule 1 (ICAM) (ab24869, abcam), PE-conjugated rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1) (ab24853, abcam), and Biotin-conjugated H-2Db (#553572, BD Pharmingen).

Techniques: Derivative Assay, Fluorescence, Imaging, Staining, Immunostaining

Journal:

Article Title: Major Histocompatibility Complex-I Expression on Embryonic Stem Cell-Derived Vascular Progenitor Cells Is Critical for Syngeneic Transplant Survival

doi: 10.1002/stem.475

Figure Lengend Snippet: Morphology, immunohistochemistry, Y-chromosome FISH, and Fluorescence-activated cell sorting (FACS) analysis following syngeneic transplantation. Tibialis muscle from female recipients was harvested 2 weeks after femoral artery ligation and male VE-CAD+MHC-I− and VE-CAD+MHC-I+ cell transplantation. H&E staining revealed increased inflammation and tissue degeneration in the VE-CAD+MHC-I− cell-transplanted group (C) compared with VE-CAD+MHC-I+ cell-transplanted group (D). In addition, increased accumulation of macrophages and T-cells was observed in the VE-CAD+MHC-I− group (G, K) as compared with the VE-CAD+MHC-I+ group (H, L). H&E staining, as well as staining for macrophages and T-cells is shown in uninjured tibialis muscle ([A], [E], [I], respectively) and at 2 weeks after femoral artery ligation with sham PBS injection ([B], [F], [J], respectively). Y-chromosome FISH staining revealed that compared with normal male ([M], Scale bar = 20 μm) and female (N) tibialis muscle, VE-CAD+MHC-I− cell-transplanted muscle (O) exhibited patchy and atypical staining, some of which was extranuclear; consistent with damaged and degenerating cells. By comparison, VE-CAD+MHC-I+ cell-transplanted muscle (P) appeared similar to the normal male muscle. (Q): Representative scatter plots from FACS analysis of DsRed positive cells (marking all transplanted cells) in the total cells extracted from ischemic muscle 2 weeks after in vivo transplantation of Bry+Flk-1+MHC-I− (Qa), Bry+Flk-1+MHC-I+ (Qb), VE-CAD+MHC-I− (Qc), and VE-CAD+MHC-I+ (Qd) cells. Abbreviations: Bry-GFP, brachyury-green fluorescent protein; DAPI, 4′,6-diamidino-2-phenylindole; FISH, fluorescent in situ hybridization; IFN-γ,interferon-γ; MHC-I, major histocompatibility complex; PBS, phosphate buffered saline; SSC, side scatter; VE-CAD, VE-cadherin.

Article Snippet: Cells were isolated, washed, and then incubated with the following primary antibodies for 60 minutes: Flk-1 (#14-5821, eBioscience, San Diego, CA, http://www.ebio-science.com/ ), VE-CAD (#555289, BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/index_us.shtml ), PE-conjugated anti-MHC-II antibody (#130-091-368, Miltenyi Biotec, Auburn CA), PE-conjugated rat anti-mouse inter-cellular adhesion molecule 1 (ICAM) (ab24869, abcam), PE-conjugated rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1) (ab24853, abcam), and Biotin-conjugated H-2Db (#553572, BD Pharmingen).

Techniques: Immunohistochemistry, Fluorescence, FACS, Transplantation Assay, Ligation, Staining, Injection, In Vivo, In Situ Hybridization

Journal:

Article Title: Major Histocompatibility Complex-I Expression on Embryonic Stem Cell-Derived Vascular Progenitor Cells Is Critical for Syngeneic Transplant Survival

doi: 10.1002/stem.475

Figure Lengend Snippet: MHC-I expression by embryonic stem cell-derived cells and subsequent NK cell attack plays a major fate-determining role following syngeneic transplantation. (A): Immunohistochemistry staining using anti-NKp46/NCR1 antibody to identify NK cells 2 weeks after donor cell transplantation into ischemic limbs that demonstrated that VE-CAD+MHC-I− cells attracted significantly more infiltrating NK cells than VE-CAD+MHC-I+ cells or sham PBS injection. Scale bar = 20 μm. (B): Infiltrating NK cells were quantitated by cell counting after immunohistochemistry staining. Statistical comparisons are shown for VE-CAD+MHC-I− versus VE-CAD+MHC-I+ cell-injected hindlimbs (****, p < .0001). (C): 51Chromium release assay; E:T ratio represents the ratio of effector NK cells to target cells. This assay revealed that target VE-CAD+MHC-I− and MHC-I− control cells (YAC-1 cells, H-2Db−) are sensitive to NK cell attack, whereas VE-CAD+MHC-I+ and MHC-I+ control cells (Concanavalin A-treated cells, H-2Db+) were resistant to effector NK cell attack. (D): In vivo NK cell depletion assay using anti-NK cell antibody, with fluorescence-activated cell sorting (FACS) performed on digested hindlimbs 2 weeks after cell administration. Control mice receiving PBS (Da) showed negligible survival of transplanted VE-CAD+MHC-I− cells (0.97% ± 0.38% of total single cells in tibialis muscle). In the anti-NK cell antibody-treated recipients (Db), 5.37% ± 0.41% of total single cells in tibialis muscle were donor derived (p < .0005 vs. PBS). Abbreviations: Co, control; E, effector cells; MHC-I, major histocompatibility complex; NK, natural killer cells; PBS, phosphate buffered saline; SSC, side scatter; T, target cells; VE-CAD, VE-cadherin; VEGF, vascular endothelial growth factor.

Article Snippet: Cells were isolated, washed, and then incubated with the following primary antibodies for 60 minutes: Flk-1 (#14-5821, eBioscience, San Diego, CA, http://www.ebio-science.com/ ), VE-CAD (#555289, BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/index_us.shtml ), PE-conjugated anti-MHC-II antibody (#130-091-368, Miltenyi Biotec, Auburn CA), PE-conjugated rat anti-mouse inter-cellular adhesion molecule 1 (ICAM) (ab24869, abcam), PE-conjugated rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1) (ab24853, abcam), and Biotin-conjugated H-2Db (#553572, BD Pharmingen).

Techniques: Expressing, Derivative Assay, Transplantation Assay, Immunohistochemistry, Staining, Injection, Cell Counting, Release Assay, In Vivo, Depletion Assay, Fluorescence, FACS

Journal:

Article Title: Major Histocompatibility Complex-I Expression on Embryonic Stem Cell-Derived Vascular Progenitor Cells Is Critical for Syngeneic Transplant Survival

doi: 10.1002/stem.475

Figure Lengend Snippet: Continued survival, proliferation, and differentiation of MHC-I+ ESC-derived progenitors after syngeneic transplantation. (A): The percentage of transplanted cells (and/or their progeny) in total single cells extracted from tibialis muscles at 2 and 4 weeks after transplantation in our ischemic hindlimb model was assessed by fluorescence-activated cell sorting (FACS) analysis. Statistical comparisons are shown between identical time-points for hindlimbs injected with Bry+Flk-1+MHC-I− versus Bry+Flk-1+MHC-I+ cells, and for hindlimbs injected with VE-CAD+MHC-I− cells versus both VE-CAD+MHC-I+ cells and VE-CAD+MHC-I− cells with NKAB (***, p < .005, ****, p < .0001, n = 7). No significant differences were found within cell groups at the two versus 4 week time-points. (B): Immunohistochemistry double staining was performed for DsRed (green) and either CD31 (red) or α-SMA (red) at 2 weeks post-transplantation. Scale bar = 10 μm. Colocalization of DsRed staining was frequently observed in association with positive staining for CD31, or α-SMA in VE-CAD+MHC-I+ cell-injected groups. (C): Immunohistochemistry double staining was performed for Y-chromosome and either α-SMA or CD31 2 weeks after transplantation of Bry+Flk-1+MHC-I+ (i)orVE-CAD+MHC-I+ (ii) cells into ischemic hindlimbs. Colocalization of Y-chromosome staining was frequently observed in association with positive staining for α-SMA or CD31. Scale bar = 20 μm. (D): Summary of FACS analysis for DsRed positive cells (marking all transplanted cells) coexpressing either CD31 or Sm22 in the total cells extracted from ischemic muscle at 2 and 4 weeks after in vivo administration of Bry+Flk-1+MHC-I+ cells, VE-CAD+MHC-I+ cells, or VE-CAD+MHC-I− cells with NKAB. Statistical comparisons are shown at identical time-points for Bry+Flk-1+MHC-I+ cells versus VE-CAD+MHC-I+ cells and VE-CAD+MHC-I− cells with NKAB (*, p < .05; ***, p < .005; ****, p < .0001). Abbreviations: a-SMA, alpha-smooth muscle actin; Bry-GFP, brachyury-green fluorescent protein; ESC, embryonic stem cell; MHC-I, major histocompatibility complex; NKAB, NK cell blocking antibody; VE-CAD, VE-cadherin; VEGF, vascular endothelial growth factor.

Article Snippet: Cells were isolated, washed, and then incubated with the following primary antibodies for 60 minutes: Flk-1 (#14-5821, eBioscience, San Diego, CA, http://www.ebio-science.com/ ), VE-CAD (#555289, BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com/index_us.shtml ), PE-conjugated anti-MHC-II antibody (#130-091-368, Miltenyi Biotec, Auburn CA), PE-conjugated rat anti-mouse inter-cellular adhesion molecule 1 (ICAM) (ab24869, abcam), PE-conjugated rat anti-mouse vascular cell adhesion molecule-1 (VCAM-1) (ab24853, abcam), and Biotin-conjugated H-2Db (#553572, BD Pharmingen).

Techniques: Derivative Assay, Transplantation Assay, Fluorescence, FACS, Injection, Immunohistochemistry, Double Staining, Staining, In Vivo, Blocking Assay