mesenchymal stem cell growth medium  (PromoCell)

Journal: bioRxiv

Article Title: Physiological re-replication during human stem cell differentiation

doi: 10.64898/2026.02.27.708451

Figure Lengend Snippet: Overview and time scale of re-replications experiments Mesenchymal stem cells were differentiated into osteoblasts, adipocytes, chondrocytes, and neurons, while myoblasts were differentiated into myotubes. Two experimental strategies were applied to detect re-replication during differentiation (A). For Rerep-seq the thymidine-analogue BrdU was added to the culture media. In the schematic representation of the re-replication bubble, yellow asterisks indicate BrdU integration. The black DNA strand represents parental DNA, the red strand corresponds to DNA from the first round of replication, the green strand to DNA from the second round, and the turquoise strand to DNA from the third round of replication. As described by Menzel et al. , UVA photolyzes incorporated BrdU converting it to Uracil. Subsequently, UDG excises the Uracil, generating an abasic site, while AP1 introduces single strand breaks at these positions. A representative agarose gel image demonstrates the effects of UVA, UDG, and AP1 treatment on DNA from chondrogenic differentiated hMSCs cultured with and without BrdU supplementation. For fiber-combing thymidine-analogues IdU and CldU were subsequently added to culture media. High molecular weight DNA was used for fiber-combing and further evaluated for fibers with simultaneous IdU and CldU incorporation. Time scale shows an overview on start and end points of all differentiation experiments (B). Yellow rectangles highlight timeframes where re-replication was detected with fiber-combing. Black arrows point on likely re-replication start points during the differentiation process. The figure was created using BioRender.

Article Snippet: The cells were seeded at a density of 100,000/25 cm 2 flask with Mesenchymal Stem cell Growth Medium (PromoCell) and expanded for 1 passage before induction of differentiation.

Techniques: Agarose Gel Electrophoresis, Cell Culture, Analogues, High Molecular Weight

Journal: bioRxiv

Article Title: Physiological re-replication during human stem cell differentiation

doi: 10.64898/2026.02.27.708451

Figure Lengend Snippet: Analysis of extranuclear DNA during differentiation Mesenchymal stem cells were grown on glass slides with corresponding differentiation media and supplemented with thymidine-analogue EdU during timeframes of re-replication determined before. EdU incorporation is detected with red fluorescence (Alexa-Fluor-594) and DNA was counterstained with Hoechst33342 in blue. An example of osteogenic differentiated hMSCs is shown and three enlarged views point to extranuclear spots and clusters of DNAs that were microdissected (A). In total 1200-1600 extranuclear DNAs were collected for further DNA isolation and sequencing for each differentiation experiment. During adipogenic differentiation extranuclear DNA was detectable after 3d and 4d and during osteogenic differentiation after 4d of differentiation. Results of sequencing analysis of extranuclear DNA were displayed using IGV with data range on the y-axis adjusted for all extranuclear DNA reads. For each differentiation extranuclear DNA sequencing (dark blue) is displayed above the corresponding Rerep-seq results (green). Representative examples for gene regions on chromosome 12 (B) and 7 (C) revealed that extranuclear DNA overlaps with re-replicated DNA regions.

Article Snippet: The cells were seeded at a density of 100,000/25 cm 2 flask with Mesenchymal Stem cell Growth Medium (PromoCell) and expanded for 1 passage before induction of differentiation.

Techniques: Fluorescence, DNA Extraction, Sequencing, DNA Sequencing

Journal: bioRxiv

Article Title: Physiological re-replication during human stem cell differentiation

doi: 10.64898/2026.02.27.708451

Figure Lengend Snippet: Asymmetric re-replication Fiber-combing experiments revealed several replication forks with varying combinations of replication and/or re-replication events. Representative examples shown, derived from neurogenic (A, B) and adipogenic (C) differentiated mesenchymal stem cells. Re-replication on two strands of the replication fork but differing in length between strands (A), re-replication only on one strand with no re-replication on the other strand of the replication fork (B) and re-replication only on one strand showing no detectable replication at the other strand of the replication fork (C). DNA is shown in blue (false color YOYO-stain) and thymidine-analogue detection is shown in red for IdU and green for CldU. Re-replicated fiber tracks appear as yellow fluorescence. Scale bars represent 20 µm = 40kb.

Article Snippet: The cells were seeded at a density of 100,000/25 cm 2 flask with Mesenchymal Stem cell Growth Medium (PromoCell) and expanded for 1 passage before induction of differentiation.

Techniques: Derivative Assay, Staining, Fluorescence

Journal: ACS Biomaterials Science & Engineering

Article Title: Fabrication of Multiscale, Multidirectional Orientated Collagen Hydrogels with Guided Cell Alignment Using Fluidics and a Three-Dimensional Printing

doi: 10.1021/acsbiomaterials.4c02156

Figure Lengend Snippet: Fluorescence microscopic observation of collagen models with MSCs after 3 days culture. The models of WM-WF (a–c, m), WM- NF (d–f, n), NM-WF (g–i, o), and NM-NF (j–l, p) are shown. Fluorescence images staining collagen with antitype I collagen antibody (green, a, d, g, and j), nuclei with DAPI (blue, b, e, h, and k), and cytoskeletal protein F-actin with rhodamine-phalloidin (red, c, f, i, and l) after 3 days of culture. Scale bars = 200 μm. The flow direction coincides with the horizontal direction of the fluorescence image (90°); The angle of F-actin distribution was plotted at the range 0–180° (m, n, o, and p).

Article Snippet: Mouse embryonic fibroblast NIH3T3 cells (American Type Culture Collection [ATCC], Virginia, USA) and primary human bone marrow-derived mesenchymal stem cells (MSCs; Promocell, Heidelberg, Germany) were cultured in Dulbecco’s modified Eagle’s medium (D-MEM; Nacalai Tesque, Inc.) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc.), and 1% of penicillin (100 U/mL)/streptomycin (100 μg/mL) solution (Nacalai Tesque, Inc.).

Techniques: Fluorescence, Staining