Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Determination of efficacy of C2C12 differentiation in conjunction with the exposure to ligand combinations. C2C12s were differentiated for 7 days and treated with combination ligands of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL for seven additional days. ( A ) Fusion index was calculated from total myotube nuclei vs. total nuclei ( n = 16, mean + SD). ( B ) Multinucleation of C2C12 myotubes were quantified ( n = 11, mean + SD). ( C ) Nuclear density was evaluated from nuclear count per field of 5x microscopy ( n = 4, mean + SD). ( D ) C2C12 exposed to ligand combinations were stained to express nuclear MYOD1 ( n = 6, mean + SD). ( E ) Cells were stained with Ki67, and where similarly quantified based on average total nuclear count ( n = 6, mean + SD). ( F ) ACTN2 and Ki67 immunostaining of control cells. ( G ) Cells exposed to GTF showed decreases in fusion index and myonucleation levels, although no change in nuclear density and Ki67+ expression was detected. ( H ) GTIF supplementation significantly reduced skeletal muscle differentiation parameters fusion index and multinucleation, in addition to decreasing average nuclear density. ( I ) Control C2C12s expressing nuclear MYOD1. ( J ) GTF treatment greatly reduced nuclear fusion and showed limited differentiation capacity while expressing comparable levels of nuclear MYOD1. ( K ) Exposure of C2C12s to GTIF combination significantly inhibited skeletal muscle differentiation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Microscopy, Staining, Immunostaining, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Effect of ligand combination exposure on differentiation of skeletal muscle cells derived from tHFs. Cells were transduced with MYOD1 fragments and induced to express the skeletal muscle phenotype via the induction of doxycycline and SB431542 over a 7-day period. Ligand combinations of GDF11 (G), TMSB4X (T), IL6 (I), and TNF-α (F) at 10 ng/mL were introduced for an additional week, and SB and Dox administration was discontinued. Skeletal muscle cells were fixed and stained on day 14 and characterized by various differentiation and proliferation parameters from 5× microscopy. ( A ) Fusion index of tHFs was evaluated by determining the ratio of myotube nuclei vs total nuclear count ( n = 16, mean + SD). ( B ) Cellular multinucleation was quantified to assess tHF development of differentiation ( n = 22, mean + SD). ( C ) Nuclear density was similarly assessed by quantifying nuclear count per field ( n = 4, mean + SD). ( D ) Nuclear MYOD1 was quantified ( n = 6, mean + SD). ( E ) Ki67 nuclei were also assessed with a nuclear count ( n = 6, mean + SD). ( F ) Control tHF myotubes were immunostained with ACTN2 and Ki67. ( G ) IL6 and TNF-α combination demonstrated significant decrease in differentiation parameters fusion index, multinucleation, myotube length, and diameter , although Ki67+ expression had increased. ( H ) Exposure of tHFs to combined GDF11, TMSB4X, IL6, and TNF-α showed similar results, however nuclear Ki67 expression was unchanged. ( I ) Untreated tHFs with ACTN2 and MYOD1 nuclear stains. ( J ) Cells treated with IF showed a decrease in MYOD1 nuclear expression. ( K ) Additionally, GDF11, TMSB4X, and IL6 exposure yielded similar results with respect to MYOD1+. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Derivative Assay, Transduction, Staining, Microscopy, Control, Expressing

Journal: Biology

Article Title: Transdifferentiation of Human Fibroblasts into Skeletal Muscle Cells: Optimization and Assembly into Engineered Tissue Constructs through Biological Ligands

doi: 10.3390/biology10060539

Figure Lengend Snippet: Skeletal muscle tissues were engineered from a composite fibrin/Matrigel hydrogel mixture with mouse skeletal myoblasts C2C12s, and subject to 10 ng/mL biological ligands. C2C12s were encapsulated and differentiated in a fibrin-based hybrid hydrogel over a 7-day period, 10 ng/mL biological ligands GDF11, TMSB4X, IL6 or TNF-α were administered after a week of tissue plating. ( A ) Immunohistochemical staining of C2C12 skeletal muscle constructs with ACTN2 and DAPI, demonstrated high cellular density. ( B ) Skeletal myotubes increased compactness and alignment towards central pillar regions where tensile force is maximal ( C ) Structural organization of C2C12s at pillar regions appeared disrupted due to gel contraction. ( D ) Cross-striated, multinucleated skeletal muscle form condensed tissues as demonstrated with high magnification 60× confocal microscopy. ( E ) Myotube diameter (µm) was not affected by one-week exposure to 10 ng/mL ligands. ( n > 32, mean + SD). ( F ) Nuclear density of skeletal muscle C2C12s within tissue were not impacted with ligand administration. ( n = 6, mean + SD).

Article Snippet: Proteins utilized included recombinant human Follistatin (Fs, 669-FO-025), recombinant human Myostatin (GDF8, 788-G8-010) or Growth Differentiation factor (GDF8), recombinant human basic Fibroblast Growth Factor 2 (FGF2, 233-FB-025), recombinant human GDF11 (1958-GD-010), recombinant human GDF15 (957-GD-025/CF), recombinant human Bone Morphogenetic Protein 4 (BMP4, 314-BP-010/CF), recombinant human BMP7 (354-BP-010), recombinant human Growth Hormone (hGH, 1067-GH-025), recombinant human Interleukin 6 (IL6, 206-IL-010), recombinant human Tumor Necrosis Factor Alpha (TNF-α, 210-TA-005) (All R&D Systems), and Thymosin β (TOCRIS, 3390).

Techniques: Immunohistochemical staining, Staining, Construct, Confocal Microscopy

Journal: Endocrinology

Article Title: Reduced Circulating GDF11 Is Unlikely Responsible for Age-Dependent Changes in Mouse Heart, Muscle, and Brain.

doi: 10.1210/en.2015-1628

Figure Lengend Snippet: Figure 1. Circulating myostatin levels decrease in aging mice. A, A myostatin standard curve was generated in the absence or presence of recombinant GDF11. Serum myostatin levels were quantified in 3-month-old wild-type and mstn/ male mice (n 5/group), again in the absence or presence exogenous addition of GDF11 (B, P .05 indicated by different letters) and in three 28 month-old wild-type mice of both sexes (C and D). Significant differences were determined by a regression analysis (C) and by a Student t test (D).

Article Snippet: Assay validation of the R&D System myostatin ELISA (ELISA, catalog No. DGDF80) was performed by running a standard curve in the absence or presence of 2 ng/mL GDF11, also acquired from R&D Systems (catalog No. 1958-GD-010).

Techniques: Generated, Recombinant

Journal: PLoS Biology

Article Title: Cell Lineages and the Logic of Proliferative Control

doi: 10.1371/journal.pbio.1000015

Figure Lengend Snippet: OE explants were cultured in various doses of GDF11. At 12 h, BrdU was added for 2 h and then washed out. Explants were fixed at various times after BrdU addition and immunostained for BrdU and NCAM expression. (A–I) Cultures grown in GDF11 concentrations of 0 (A, D, and G), 0.5 (B, E, and H), and 10 (C, F, and I) ng/ml, fixed 18 h after BrdU addition (previous studies have shown that 18 h is sufficient time for INP progeny that become ORNs to express NCAM ). NCAM immunofluorescence (green) is shown in (A–C); BrdU immunofluorescence (red) in (D–F); merged images in (G–I). Arrowheads point to examples of BrdU + /NCAM − cells; arrows point to examples of BrdU + /NCAM + cells. (J) Percentage of BrdU + cells migrating out of OE explants that had differentiated (acquired NCAM immunoreactivity) by 18 h (black line) or 36 h (blue line), as a function of GDF11 dose. Low doses of GDF11 increase the proportion of INP progeny that differentiate (i.e., p 1 decreases). At high dose, the effect reverses, with the NCAM + fraction falling to near zero at 18 h, but recovering at 36 h. These data are consistent with a slowing of the cell cycle ( v 1 ) such that 18 h is not long enough to produce NCAM + offspring (but 36 h is). This interpretation is consistent with a previous demonstration that high doses of GDF11 reversibly arrest the INP cell cycle . (K) Simulation of the experiment in (J) by a model in which GDF11 affects both p 1 and v 1 . Parameters used in the model are consistent with measured proportions of ORNs, INPs, and Mash1 + / Sox2 + cells, as well as experimental data on the effects of GDF11 on BrdU pulse-labeling by INPs [ , , ].

Article Snippet: OE explants were prepared as previously described [ ] and cultured with 10 ng/ml recombinant FGF2 and varying concentrations of GDF11 (PeproTech).

Techniques: Cell Culture, Expressing, Immunofluorescence, Labeling

Journal: PLoS Biology

Article Title: Cell Lineages and the Logic of Proliferative Control

doi: 10.1371/journal.pbio.1000015

Figure Lengend Snippet: (A) Simulations of the model in D were carried out for 20,000 randomly chosen sets of parameters ( – , section 8). To simulate regeneration following a loss of terminal-stage cells, numbers of ORNs were set to zero, whereas numbers of stem cells and transit-amplifying cells (INPs) were set to their steady state values. For each parameter set, the time it took for ORN numbers to return to and remain within 20% of their steady state values was taken as an objective measure of regeneration time, and cases with very long regeneration times (>29 transit-amplifying cell cycle lengths) are not shown (see – ). Next, the time that would have been required to generate the same number of ORNs, from the same initial conditions but in the absence of feedback, was calculated. Finally, the ratio of the two regeneration times (with and without feedback) was considered to be the fold improvement in regeneration speed due to feedback. For each parameter set, this was plotted against the sensitivity of the steady state solution to variation in either the initial number of stem cells, the stem cell cycle time, or the normal lifetime of ORNs (all three sensitivities are equal). The data show that only those parameter sets that do not support a robust ORN steady state (abscissa values >0.4) show substantial improvement in regeneration speed (ordinate values >2). (B) Simulated regeneration for the set of parameters in (A) that showed the greatest improvement in regeneration consistent with sensitivity to parameters remaining below 0.4 (this corresponds to a 32% change in steady state values for a 2-fold change in parameters). As in , the blue curve denotes ORN numbers, the red curve shows INPs, and the dashed line shows the time course over which regeneration would proceed in the absence of feedback. The light-blue zone denotes the range of cell numbers within 20% of the steady state value for ORNs. (C) Simulated regeneration for the parameters used in C, but starting from two different initial conditions. The solid blue curve shows the dynamics of ORN recovery after complete removal of existing ORNs; the solid gray curve illustrates the predicted rate of recovery in the absence of feedback. The dashed blue and gray curves present corresponding simulations where ORN numbers were initially depleted only 75%, rather than completely. Under these conditions, nearly all improvement in regeneration is lost. (D) To quantify the effect of initial conditions on regeneration speed, a ratio was defined (“speed ratio”) that indicates how much faster (or slower) regeneration from 75% ORN depletion is than regeneration from 100% depletion. In the absence of feedback, this ratio should have a value of approximately 1.22 (regeneration from partial depletion should take slightly less time than regeneration from total depletion). This ratio was calculated for each of the random cases shown in (A), and the results were plotted against the fold improvement in regeneration speed (from [A]). The abscissa is drawn at an ordinate value of 1.22. The plot shows that the more one gains in regeneration speed from 100% depletion, the more one sacrifices in regeneration speed from 75% depletion. (E) Negative feedback effects of activin and GDF11 (shown diagrammatically in red) can be modeled by multiplying the replication probabilities and cell division rates of stem cells and INPs, respectively, by decreasing functions of ORN numbers (χ 2 ). In this case, Hill functions are used, with parameters g , h , j , and k representing the feedback gains, and n the Hill coefficient. (F) Example of a case with both activin and GDF11 feedback. Notice that now, regeneration from initial conditions of 75% ORN depletion is nearly as fast as regeneration from 100% ORN depletion (compare with [C]). Parameters for this case are: p 0 = 0.507, p 1 = 0.546, d / v 1 = 0.0116, v 0 / v 1 = 0.965, g = 1.258, h = 1.03, j = 0.0394, and k = 1.683 (and the ordinate axis has been scaled for easier comparison with [C]). In (B), (C), and (F), time is expressed in units of ln 2/ v 1 .

Article Snippet: OE explants were prepared as previously described [ ] and cultured with 10 ng/ml recombinant FGF2 and varying concentrations of GDF11 (PeproTech).

Techniques: Comparison

Journal: PLoS Biology

Article Title: Cell Lineages and the Logic of Proliferative Control

doi: 10.1371/journal.pbio.1000015

Figure Lengend Snippet: (A and B) Polypeptides secreted into the intercellular space of an epithelium are removed by two processes: diffusion into underlying connective tissue (stroma) and degradation within the epithelium. Given a molecule's rate of production, its diffusivity, its rate of uptake and degradation, and the geometry of the epithelium, one may calculate its concentration, at steady state, at every location within the epithelium. Here, such calculations are shown graphically, for epithelia of different thicknesses (in each picture, the epithelium is oriented with the apical surface at the top). Epithelial thickness (“height”) is scaled according to the decay length of the molecule of interest. The shading in each picture depicts the concentration of the secreted molecule, with black representing the limiting concentration that would be achieved in an epithelium of infinite thickness. In (A), the degradation capacity of the stroma is set at a relatively low value, one-tenth of that in the epithelium. In this case, intraepithelial concentrations of secreted molecules plateau while the epithelium is very thin. In (B), the degradation capacity of the stroma is ten times of that in the epithelium, so that few molecules that enter the stroma escape undegraded. Now, there is a large (and more physiological) range of epithelial thickness over which the concentrations of secreted molecules change appreciably with tissue size. This is particularly true near the basal surface of the epithelium (see also Figures S27 and S28 in – ). (C) Follistatin (FST), a molecule that binds GDF11 and activin essentially irreversibly, is present at high levels in the basal lamina (arrow) and stroma (asterisk) beneath the embryonic day 13 OE. Association of FST with basal laminae is consistent with its affinity for extracellular matrix components . Scale bar represents 100 μm. (D and E) INPs (visualized with Ngn1 in situ hybridization) become progressively localized to the basal surface of the OE over the course of development. (D) = embryonic day 12.5; (E) = embryonic day 18.5. nc = nasal cavity. Scale bar in (E) represents 100 μm.

Article Snippet: OE explants were prepared as previously described [ ] and cultured with 10 ng/ml recombinant FGF2 and varying concentrations of GDF11 (PeproTech).

Techniques: Diffusion-based Assay, Concentration Assay, In Situ Hybridization

Journal: Kidney & Blood Pressure Research

Article Title: GDF11 Improves Ischemia-Reperfusion-Induced Acute Kidney Injury via Regulating Macrophage M1/M2 Polarization

doi: 10.1159/000529444

Figure Lengend Snippet: Effect of AKI induced by I/R on GDF11 expression. a The protein expression of GDF11 in mouse kidney tissue was detected by IHC. The brown color in IHC represented the positive staining of GDF11. b The protein expression of GDF11 in mouse kidney tissue was detected by WB. c The mRNA level of GDF11 in kidney tissues was detected by q-PCR. Normal, normal group; Sham, sham group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R7D, 7 days after I/R. * p < 0.05, ** p < 0.01; NS, p > 0.05 compared with the sham group.

Article Snippet: Recombinant GDF11 was purchased from Abnova (Taiwan, China).

Techniques: Expressing, Staining

Journal: Kidney & Blood Pressure Research

Article Title: GDF11 Improves Ischemia-Reperfusion-Induced Acute Kidney Injury via Regulating Macrophage M1/M2 Polarization

doi: 10.1159/000529444

Figure Lengend Snippet: Flow cytometry assay of the effect of GDF11 on M1/M2 polarization of macrophages in vitro.

Article Snippet: Recombinant GDF11 was purchased from Abnova (Taiwan, China).

Techniques: Flow Cytometry, In Vitro

Journal: Kidney & Blood Pressure Research

Article Title: GDF11 Improves Ischemia-Reperfusion-Induced Acute Kidney Injury via Regulating Macrophage M1/M2 Polarization

doi: 10.1159/000529444

Figure Lengend Snippet: Effect of GDF11 on the serum levels of cytokines after I/R treatment. The serum levels of IL-6 ( a ), IL-1β ( b ), TNF-α ( c ), IL-4 ( d ), IL-10 ( e ), and TGF-β ( f ) were measured. Normal, normal group; Sham, sham group; I/R 1D, 1 day after I/R; I/R 3D, 3 days after I/R; I/R 7D, 7 days after I/R. * p < 0.05, ** p < 0.01; NS, p > 0.05 compared with the sham group.

Article Snippet: Recombinant GDF11 was purchased from Abnova (Taiwan, China).

Techniques:

Journal: Kidney & Blood Pressure Research

Article Title: GDF11 Improves Ischemia-Reperfusion-Induced Acute Kidney Injury via Regulating Macrophage M1/M2 Polarization

doi: 10.1159/000529444

Figure Lengend Snippet: In vivo study of the effect of GDF11 treatment on M1/M2 polarization of macrophages in I/R mouse model.

Article Snippet: Recombinant GDF11 was purchased from Abnova (Taiwan, China).

Techniques: In Vivo