Journal: Cell Transplantation

Article Title: c-Casitas b-Lineage Lymphoma Downregulation Improves the Ability of Long-term Cultured Mesenchymal Stem Cells for Promoting Angiogenesis and Diabetic Wound Healing

doi: 10.1177/0963689721989605

Figure Lengend Snippet: The phosphorylation level of Akt was decreased in long-term cultured MSCs and increased by treating with c-Cbl LNA Gapmers. (A) Representative immunoblots and histogram analysis of Y731c-Cbl/c-Cbl and S473-Akt/Akt in P3, P6, and P10 MSCs ( n = 3). Error bars represented the mean ± SE * P < 0.05 compared with the P3 group and # P < 0.05 versus between the indicated groups. (B) Representative immunoblots and histogram analysis of c-Cbl and S473-Akt/Akt in P10, P10 + SCR, and P10 + c-Cbl KD group cells ( n = 3). Error bars represented the mean ± SE. * P < 0.05 compared with the P10 group and # P < 0.05 versus between the indicated groups. c-Cbl: c-Casitas b-lineage lymphoma; LNA Gapmers: locked nucleic acid–modified antisense oligonucleotide gapmers; MSC: mesenchymal stem cell; SE: standard error.

Article Snippet: BM-MSCs were cultured in MSCs complete medium (Cyagen Biosciences) and HUVECs were cultured in endothelial cell (EC) complete medium (Cyagen Biosciences).

Techniques: Phospho-proteomics, Cell Culture, Western Blot, Modification

Journal: Cell Transplantation

Article Title: c-Casitas b-Lineage Lymphoma Downregulation Improves the Ability of Long-term Cultured Mesenchymal Stem Cells for Promoting Angiogenesis and Diabetic Wound Healing

doi: 10.1177/0963689721989605

Figure Lengend Snippet: The migration and angiogenic factors paracrine activity were decreased in long-term cultured MSCs and increased by treating with c-Cbl LNA Gapmers. (A) HUVECs were suspended in EC complete medium (PC) or vehicle (NC)-, P3 MSCs-, P10 MSCs-P10 + scramble LNA Gapmers (P10 + SCR) MSCs-, P10 + c-Cbl LNA Gapmers (P10 + c-Cbl KD) MSCs-conditioned EC basal medium supplemented with 0.75% FBS, and incubated for 8 h (original magnification, 10×) ( n = 3). The tube length, nodes, and meshes in different groups counted in three fields per well were statistically analyzed by histogram analysis. (B) The VEGFA, HGF, and bFGF level in the conditioned media were assessed by ELISA assay ( n = 3). (C) The representative picture and histogram analysis of the stained MSCs migrated to the bottom surface of the upper chamber with 10× magnification in each group ( n = 3). Error bars represent the mean ± SE. # P < 0.05 compared with the NC group and * P < 0.05 versus between the indicated groups. bFGF: basic fibroblast growth factor; c-Cbl: c-Casitas b-lineage lymphoma; EC: endothelial cell; HGF: hepatocyte growth factor; HUVEC: human umbilical vein endothelial cell; LNA Gapmers: locked nucleic acid–modified antisense oligonucleotide gapmers; MSC: mesenchymal stem cell; NC: negative control; PC: positive control; SE: standard error; VEGFA: vascular endothelial growth factor A.

Article Snippet: BM-MSCs were cultured in MSCs complete medium (Cyagen Biosciences) and HUVECs were cultured in endothelial cell (EC) complete medium (Cyagen Biosciences).

Techniques: Migration, Activity Assay, Cell Culture, Incubation, Enzyme-linked Immunosorbent Assay, Staining, Modification, Negative Control, Positive Control

Journal: Cell Transplantation

Article Title: c-Casitas b-Lineage Lymphoma Downregulation Improves the Ability of Long-term Cultured Mesenchymal Stem Cells for Promoting Angiogenesis and Diabetic Wound Healing

doi: 10.1177/0963689721989605

Figure Lengend Snippet: The effect of c-Cbl downregulation on cellular proliferation and senescence of long-term cultured MSCs and the timeline for diabetic wound observation and sampling. (A) The representative picture of SA-β-gal stained BM-MSCs in different groups. (B) The histogram analysis of SA-β-gal-positive senescent MSCs. Results are expressed as means ± SE. * P < 0.05 versus between the indicated groups. (C) The cellular proliferation activity was assessed by CCK-8 assay. All data are represented as means ± SE. # P < 0.05 compared with the P10 + c-Cbl KD group and * P < 0.05 compared with P3 group. (D) After wound induction and injection of MSCs, wounds were photographed at the indicated time points. On days 7 and 14, the wound tissue was harvested and fixed with 10% formalin. c-Cbl: c-Casitas b-lineage lymphoma; CCK-8: Cell Counting Kit-8; LNA Gapmers: locked nucleic acid–modified antisense oligonucleotide gapmers; MSC: mesenchymal stem cell; SA-β-Gal: senescence associated β-galactosidase; SE: standard error.

Article Snippet: BM-MSCs were cultured in MSCs complete medium (Cyagen Biosciences) and HUVECs were cultured in endothelial cell (EC) complete medium (Cyagen Biosciences).

Techniques: Cell Culture, Sampling, Staining, Activity Assay, CCK-8 Assay, Injection, Cell Counting, Modification

Journal: Cell Transplantation

Article Title: c-Casitas b-Lineage Lymphoma Downregulation Improves the Ability of Long-term Cultured Mesenchymal Stem Cells for Promoting Angiogenesis and Diabetic Wound Healing

doi: 10.1177/0963689721989605

Figure Lengend Snippet: c-Cbl downregulation increased the effect of long-term cultured MSCs on promoting VEGFA expression and diabetic rat wound angiogenesis. (A) The staining of CD31 and VEGFA in diabetic wounds was detected by immunohistochemistry on the 7th and 14th day after surgery (original magnification, 10× and 40×) ( n = 4). (B) Microvessels were showed as positive CD31 staining and typical round or oval structure. (C) Fluorescent intensity of VEGFA was quantified by densitometry and normalized to the control group. The values are presented as means ± SE. # P < 0.05 versus between the indicated groups. c-Cbl: c-Casitas b-lineage lymphoma; MSC: mesenchymal stem cell; SE: standard error; VEGFA: vascular endothelial growth factor A.

Article Snippet: BM-MSCs were cultured in MSCs complete medium (Cyagen Biosciences) and HUVECs were cultured in endothelial cell (EC) complete medium (Cyagen Biosciences).

Techniques: Cell Culture, Expressing, Staining, Immunohistochemistry, Control

Journal: Cell Transplantation

Article Title: c-Casitas b-Lineage Lymphoma Downregulation Improves the Ability of Long-term Cultured Mesenchymal Stem Cells for Promoting Angiogenesis and Diabetic Wound Healing

doi: 10.1177/0963689721989605

Figure Lengend Snippet: c-Cbl downregulation increased the effect of long-term cultured MSCs on wound healing in diabetic rats. (A) General view of wound healing in the diabetic rat model treated with PBS (control), P10 MSCs + scramble LNA Gapmers (P10 + SCR), and P10 MSCs + c-Cbl LNA Gapmers (P10 + c-Cbl KD) at 0, 3, 7, and 14 days postoperation. At the indicated times, the wound closure rate of all treatment groups was analyzed and represented in the linear diagram ( n = 8). The values are presented as means ± SE. * P < 0.05 compared with control and # P < 0.05 compared with P10 + SCR group. (B) Representative micrographs of H&E and Masson’s staining of skin wound tissues at 14 days after the operation (original magnification, 4× and 10×) ( n = 4). (C) The histogram analysis of wound healing scores in three groups ( n = 8). The values are presented as means ± SE. * P < 0.05 versus between the indicated groups. c-Cbl: c-Casitas b-lineage lymphoma; H&E: hematoxylin and eosin; LNA Gapmers: locked nucleic acid–modified antisense oligonucleotide gapmers; MSC: mesenchymal stem cell; PBS: phosphate buffered saline; SE: standard error.

Article Snippet: BM-MSCs were cultured in MSCs complete medium (Cyagen Biosciences) and HUVECs were cultured in endothelial cell (EC) complete medium (Cyagen Biosciences).

Techniques: Cell Culture, Control, Staining, Modification, Saline

Journal: Advanced Science

Article Title: Cells‐Micropatterning Biomaterials for Immune Activation and Bone Regeneration

doi: 10.1002/advs.202200670

Figure Lengend Snippet: Cell morphology and activity of MSCs and macrophages in different multicellular patterning bioactive scaffolds. A) SEM images of MSCs and macrophages cultured in different multicellular patterning scaffolds at a ratio of 2:1 for 5 days. B) MSCs and C) macrophages proliferation in different multicellular patterning scaffolds for 2 and 5 days, respectively. Data presented as mean ± SD, n = 4 for each scaffold type, p ‐values are calculated using one‐way ANOVA with Tukey correction. * indicates significant difference, * p < 0.05, ** p < 0.01, and *** p < 0.001; ns, not statistically significant. D) CLSM 2D (left side) and 3D (right side) images of MSCs and macrophages penetrating deep into channels after incubating for 5 days. MSCs and macrophages were labeled with red and green fluorescent probes, respectively. MSCs or macrophages monocultured in the scaffolds were used as controls and named as Control‐MSC and Control‐MΦ, respectively. The two kinds of cells grew well in these multicellular patterning scaffolds, exhibiting good cell viabilities.

Article Snippet: New Zealand white rabbit bone marrow‐derived MSCs were purchased from Cyagen Biotechnology Co., Ltd. (Cyagen, Guangzhou, China) and cultured in rabbit MSCs culture medium (Cyagen, Guangzhou, China).

Techniques: Activity Assay, Cell Culture, Labeling

Journal: Advanced Science

Article Title: Cells‐Micropatterning Biomaterials for Immune Activation and Bone Regeneration

doi: 10.1002/advs.202200670

Figure Lengend Snippet: In vitro osteogenic differentiation behaviors of MSCs in different multicellular patterning bioactive scaffolds with a 2:1 ratio of MSCs to macrophages. A–C) Gene expression of BMP‐Smad, OSM, and Wnt/β‐catenin signaling pathway‐associated mediators in MSCs cultured on different multicellular patterning scaffolds for 3 days. D) The osteogenic gene expression of MSCs in different multicellular patterns on day 3. Data presented as mean ± SD, n = 4 for each scaffold type, p ‐values are calculated using one‐way ANOVA with Tukey correction, * p < 0.05, ** p < 0.01, and *** p < 0.001; ns, not statistically significant. E) Representative immunofluorescence images of BMP‐2 protein expression in MSCs grown on scaffolds with different multicellular patterns for 3 days. F) ALP staining and G) immunofluorescent staining of OCN for MSCs cultured on scaffolds with different multicellular patterns for 7 days. The Tai Chi pattern with a 2:1 ratio of MSCs to macrophages could significantly promote the osteogenic differentiation of MSCs as compared with the other multicellular patterns.

Article Snippet: New Zealand white rabbit bone marrow‐derived MSCs were purchased from Cyagen Biotechnology Co., Ltd. (Cyagen, Guangzhou, China) and cultured in rabbit MSCs culture medium (Cyagen, Guangzhou, China).

Techniques: In Vitro, Expressing, Cell Culture, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Cells‐Micropatterning Biomaterials for Immune Activation and Bone Regeneration

doi: 10.1002/advs.202200670

Figure Lengend Snippet: The modulatory effects of multicellular patterns with different cell ratios on MSC‐macrophage cross‐talk. A–D) when the ratio of MSCs to macrophages was A) 1:1, B) 1:2, C) 1:3, and D) 3:1, respectively, gene expression heatmap of immunomodulatory, angiogenic, osteogenic, and osteoclastic factors of the two kinds of cells cultured in different multicellular patterning scaffolds for 3 days via qRT‐PCR analysis. E) Gene expression heatmap of macrophages in the Tai Chi pattern with different cell ratios on day 3. F) Heatmap of osteogenic gene expression of MSCs in the Tai Chi pattern with different cell ratios on day 3. n = 4 for each scaffold type. Statistical interpretation was discussed in the Experimental Section. The different spatial distributions and cell ratios of MSCs and macrophages could trigger distinct “cross‐talk” between the two kinds of cells, thus affecting their differentiation behaviors and immunomodulatory functions.

Article Snippet: New Zealand white rabbit bone marrow‐derived MSCs were purchased from Cyagen Biotechnology Co., Ltd. (Cyagen, Guangzhou, China) and cultured in rabbit MSCs culture medium (Cyagen, Guangzhou, China).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR