Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Isolation of Placenta-Derived MSCs. (A) Illustration of the MSC isolation process. (B) Representative morphology of MSCs during isolation. (C) Differentiation of MSCs into adipocytes (FABP-4 + ) and osteocytes (osteocalcin + ). (D) Flow cytometry analysis of MSC surface markers. Negative markers include CD34, CD45, CD11b, CD79A, and HLA-DR. (E) Size distribution of MSCs cultured in T-25 flasks at passages 4 (P4), 6 (P6), and 8 (P8).

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Isolation, Derivative Assay, Flow Cytometry, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Identifying the Optimal Spheroid Diameter. (A) Representative images of MSC (P3) spheroids cultured for 3 days with varying initial cell numbers per spheroid. (B) Quantification of spheroid diameter over the 3-day culture period. (C) Representative images of individual MSCs following 2D and 3D spheroid culture. (D,E) Cell size distribution of MSCs after 2D and 3D spheroid culture. (F) Proportions of small (≤15 µm) and large (>15 µm) MSCs after 2D and 3D spheroid culture. Spheroid culture duration in (C–F) was 72 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: 3D Spheroid Culture Reduces MSC Size: Determining the Optimal Culture Duration. (A) Representative images showing morphological changes in MSC (P4) spheroids over a 7-day culture period. (B) Images of individual MSCs cultured in 2D or in 25 K-cell spheroids for varying durations. (C) Changes in the diameter of 25 K-cell MSC 3D spheroids over 7 days. (D) Comparison of mean MSC diameter in 2D versus 25 K spheroid cultures over 7 days (E,F) Size distribution of MSCs within 25 K spheroids across the 7-day culture period.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effect of Chemically Defined Medium on MSC Viability and Size in Spheroid Culture. (A) Phase-contrast and dead cell staining (red) images of MSC (P6) spheroids cultured in various media. Three representative spheroids are shown in each condition. (B) MSC size distribution in 3D cultures across different media conditions. (C,D) Anti-inflammatory capability of MSCs cultured under varying conditions. RAW-Dual™ reporter cells were stimulated with 100 ng/mL LPS and 10 ng/mL IFNγ, then treated with MSCs. Dexamethasone (Dex, 1 µg/mL) was used as a positive control. Luciferase activity (C) and mouse IL-6 (D) were measured. Spheroid culture time was 48 h.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Staining, Cell Culture, Positive Control, Luciferase, Activity Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Effects of Alternating 2D/3D Culture on MSC Size and Immunomodulatory Function. (A) MSCs were cultured in flasks for four passages, with an additional 2-day spheroid culture following each passage. Shown are the mean cell diameters immediately after 2D culture and after subsequent spheroid culture. (B) MSC diameters from P5 to P8 using conventional 2D culture versus the alternating 2D/3D method. P4 cells served as the starting population for both conditions. Diameters were measured after harvesting from 2D flasks. (C) Comparison of MSC sizes at P5–P8 between the two culture methods. (D) Comparison of doubling times at P5–P8 between the two methods. (E,F) Macrophages were activated with LPS and IFNγ, then treated with either early-passage (P4) or late-passage (P15) MSCs derived from conventional 2D culture or the alternating 2D/3D protocol. Levels of mouse IL-6 and IL-10 were measured to assess immunomodulatory effects.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Cell Culture, Comparison, Derivative Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: RGD-Modified Alginate Hydrogel Tubes (AlgTubes) for MSC Culture. (A) Schematic of alginate modification with RGD peptides. (B,C) Chemistry underlying hydrogel tube formation. (D,E) Process AlgTubes using a micro-extruder: a cell suspension and alginate solution are pumped into the central and side channels, respectively, creating coaxial core-shell flows. These are extruded through a nozzle into a CaCl 2 buffer, where Ca 2+ ions crosslink the outer alginate shell, forming hydrogel tubes instantly. (F) Illustration of growing MSCs within an AlgTube. (G,H) SEM images showing the porous structure of the AlgTubes.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Suspension

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Alternating 2D and 3D culture reduces cell size and extends the lifespan of placenta-derived mesenchymal stem cells

doi: 10.3389/fbioe.2025.1632810

Figure Lengend Snippet: Dynamic Cell Adhesion in RGD-Modified AlgTubes. (A) MSCs were processed into RGD-modified AlgTubes. (B–D) Cells adhered to the inner surface and proliferated from day 0 to day 6. (E) On day 6, free RGD peptides were added to the culture medium, leading to cell contraction within 24 h. (F) By 48 h, cells had fully detached and formed spheroids (red arrows). (G,H) Removal of free RGD peptides from the medium resulted in MSC reattachment to the AlgTube surface and resumed cell growth (blue arrows). (I–L) As a comparison, in the absence of free RGD peptides, day 6 MSCs continued to grow and eventually covered the inner surface of the hydrogel tube.

Article Snippet: MSCs were characterized using the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), which includes antibodies against the positive markers CD90, CD73, and CD105, as well as the negative markers CD45, CD34, CD11b, CD79A, and HLA-DR. Additionally, the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) was used to assess expression of the positive markers CD44, CD106, CD146, and CD166.

Techniques: Modification, Comparison

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: Generation of iPSCs and NPCs from human skin fibroblasts (NB1RGB). ( A ) To identify the PSC status of the established iPSC clone 2 (C2) from human skin fibroblasts (NB1RGB), cells were immunostained using PSC markers Nanog, SSEA4, OCT4 and KLF4. The cell nucleus was counterstained with 4′,6-diamidino-2-phenylindole (DAPI). ( B ) iPSC-like colonies were stained with alkaline phosphatase staining. ( C ) To confirm pluripotency, iPSCs were differentiated into three germ layers and stained with germ-layer markers Otx2, Brachury and Sox17 for the ectoderm, mesoderm and endoderm, respectively. ( D ) iPSCs were differentiated to neural progenitor cells (NPCs) and stained with neural stem cell markers GFAP, Nestin, Pax6, Sox1 and Sox2 and the differentiated neuron marker β-III Tubulin (Tuj1). NPCs were positive for all the NPC markers except Tuj1. Scale bar represents 25 μm or 50 μm, as indicated. iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, PSC, pluripotent stem cell = DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Staining, Marker

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: DDR after IR in iPSCs and NPCs. ( A ) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 2 Gy and fixed with 4% PFA at designated times. Cells were immunostained with 53BP1 (green) and γ-H2AX (red) antibodies. The cell nucleus was counterstained with DAPI. ( B ) Immunostained cells containing >10 53BP1- and γ-H2AX-positive foci (dot foci) were counted and graphed. ( C ) γ-H2AX apoptotic foci (pan staining) were also counted. At least 200 cells were counted and all experiments were performed three times. Scale bar represents 25 μm. Error bars represent the standard error of the mean (SEM). Welch’s (one-tailed) t -test was performed and statistical significance is indicated by asterisks ( * P < 0.05; ** P < 0.01; *** P < 0.001). DDR = DNA damage response, IR = ionizing radiation, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, PFA = paraformaldehyde, 53BP1 = p53 binding protein 1, γ-H2AX = γ-H2A histone family member X, DAPI = 4′,6-diamidino-2-phenylindole.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Irradiation, Staining, One-tailed Test, Binding Assay

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: Apoptosis detection by TUNEL assay in iPSCs. ( A ) Fibroblasts (NB1RGB) and iPSCs (NB1RGB C2) were irradiated at 2 Gy and fixed at designated times after IR. Apoptosis was detected via the TUNEL assay. ( B ) At least 200 cells were counted and all experiments were performed three times and graphed. Scale bar represents 25 μm. Error bars represent the SEM. Welch’s (one-tailed) t -test was performed and statistical significance is indicated by asterisks ( * P < 0.05). iPSCs = induced pluripotent stem cells, TUNEL = terminal deoxynucleotidyl transferase dUTP nick end labeling, SEM = standard error of the mean.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: TUNEL Assay, Irradiation, One-tailed Test

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: RNA-Seq analysis revealed transcriptional alteration in fibroblasts, iPSCs and NPCs. ( A ) Fibroblasts (NB1RGB), iPSCs (NB1RGB C2 and 201B7) and NPCs (C2) were irradiated at 5 Gy. One hour after IR, RNA samples were extracted and analyzed using the Illumina HiSeq 2500 next-generation sequencer for RNA-Seq. Raw data were quality controlled and aligned to the reference hg19. Heat maps classed as HR repair, NHEJ repair and apoptosis were obtained using R software. Red, upregulated; green, downregulated. ( B ) Differential expression patterns of each gene were represented by FPKM. Expression differences were classified according to molecular machinery components: DNA repair, cell cycle checkpoints and apoptosis. ( C ) Differential expression patterns by RNA-Seq were confirmed by immunoblotting. ATM, NBS1, CHK1, p53 and p21 antibodies were used for immunoblotting. GAPDH antibody was used as loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in and . RNA-Seq = RNA sequencing, iPSCs = induced pluripotent stem cells, NPCs = neural progenitor cells, HR = homologous recombination, NHEJ = nonhomologous end joining, FPKM = fragments per kilobase of exon per million reads mapped.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Irradiation, Software, Quantitative Proteomics, Expressing, Western Blot, Control, Homologous Recombination

Journal: Journal of Radiation Research

Article Title: Reprogramming and differentiation-dependent transcriptional alteration of DNA damage response and apoptosis genes in human induced pluripotent stem cells

doi: 10.1093/jrr/rrz057

Figure Lengend Snippet: IR sensitivity and persistent activation of DDR after IR in iPSCs . ( A ) To determine cell sensitivity to IR exposure, colony formation assay was performed. Fibroblasts (NB1RGB) and iPSCs (NB1RGBC2 and 201B7) were used. Compared with fibroblasts, iPSCs showed hypersensitivity to IR exposure. Error bars represent the SEM. ( B ) After IR exposure, cell extracts were immunoblotted with ATM, p53, serine 15 phospho-p53, KAP1 and serine 824 phospho-KAP1 antibodies. GAPDH antibody was used as a loading control. Immunoblotting data were obtained using C-digit as digital data. Full scan data of each band are provided in . DDR hyperactivation increased in a time-dependent manner in iPSCs but not in fibroblasts and NPCs. IR = ionizing radiation, DDR = DNA damage response, iPSCs = induced pluripotent stem cells, SEM = standard error of the mean, ATM = ataxia-telangiectasia-mutated, KAP1 = Kruppel-associated box domain (KRAB)-associated protein-1, GAPDH = glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: iPSCs were differentiated into the three germ layers using a Human Pluripotent Stem Cell Functional Identification Kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Colony Assay, Control, Western Blot