Macromolecular Crowding as a Tool to Screen Anti-fibrotic Drugs: The Scar-in-a-Jar System Revisited

An unsolved therapeutic problem in fibrosis is the overproduction of collagen. In order to screen the effect of anti-fibrotic drugs on collagen deposition, the Scar-in-a-Jar approach has been introduced about a decade ago. With macromolecular crowding a rapid deposition of collagen is seen, resulting in a substantial decrease in culture time, but the system has never been tested in an adequate way. We therefore have compared six different macromolecular crowders [Ficoll PM 70 (Fc70), Ficoll PM 400 (Fc400), a mixture of Ficoll 70 and 400 (Fc70/400), polyvinylpyrrolidone 40 (PVP40), polyvinylpyrrolidone 360 (PVP360), neutral dextran 670 (ND670), dextran sulfate 500 (DxS500), and carrageenan (CR)] under profibrotic conditions (addition of TGFβ1) with primary human adult dermal fibroblasts in the presence of 0.5 and 10% FBS. We found that (1) collagen deposition and myofibroblast formation was superior with 0.5% FBS, (2) DxS500 and CR results in an aberrant collagen deposition pattern, (3) ND670 does not increase collagen deposition, and (4) CR, DxS500, and Fc40/700 affected important phenotypical properties of the cells when cultured under pro-fibrotic conditions, whereas PVP40 and PVP360 did less or not. Because of viscosity problems with PVP360, we conclude that PVP40 is the most optimal crowder for the screening of anti-fibrotic drugs. Finally, the effect of various concentrations of Imatinib, Galunisertib, Omipalisib or Nintedanib on collagen deposition and myofibroblast formation was tested with PVP40 as the crowder.

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Th17 cells favor inflammatory responses while inhibiting type I collagen deposition by dermal fibroblasts: differential effects in healthy and systemic sclerosis fibroblasts

Arthritis Research & Therapy ◽

10.1186/ar4334 ◽

2013 ◽

Vol 15 (5) ◽

pp. R151 ◽

Cited By ~ 44

Author(s):

Nicolò Brembilla ◽

Elisa Montanari ◽

Marie-Elise Truchetet ◽

Elena Raschi ◽

Pierluigi Meroni ◽

...

Keyword(s):

Systemic Sclerosis ◽

Th17 Cells ◽

Type I Collagen ◽

Inflammatory Responses ◽

Dermal Fibroblasts ◽

Type I ◽

Collagen Deposition ◽

Differential Effects

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Dual inhibition by phosphodiesterase 5 and 5-HT2BInhibitor leads to near complete amelioration of fibrotic potential of human adult dermal fibroblasts isolated from a scleroderma patient

Indian Journal of Rheumatology ◽

10.4103/injr.injr_169_19 ◽

2020 ◽

Vol 0 (0) ◽

pp. 0

Author(s):

Vikas Agarwal ◽

Saurabh Chaturvedi ◽

MohitKumar Rai ◽

Harshit Singh ◽

DurgaPrasanna Misra ◽

...

Keyword(s):

Dermal Fibroblasts ◽

Human Adult ◽

Scleroderma Patient ◽

Dual Inhibition ◽

Phosphodiesterase 5

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Transdifferentiation of human adult peripheral blood T cells into neurons

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1720273115 ◽

2018 ◽

Vol 115 (25) ◽

pp. 6470-6475 ◽

Cited By ~ 33

Author(s):

Koji Tanabe ◽

Cheen Euong Ang ◽

Soham Chanda ◽

Victor Hipolito Olmos ◽

Daniel Haag ◽

...

Keyword(s):

Peripheral Blood ◽

Mononuclear Cells ◽

Ips Cells ◽

Single Step ◽

Dermal Fibroblasts ◽

Peripheral Blood Mononuclear ◽

Neuronal Markers ◽

Human Adult ◽

Adult Peripheral Blood ◽

In Cells

Human cell models for disease based on induced pluripotent stem (iPS) cells have proven to be powerful new assets for investigating disease mechanisms. New insights have been obtained studying single mutations using isogenic controls generated by gene targeting. Modeling complex, multigenetic traits using patient-derived iPS cells is much more challenging due to line-to-line variability and technical limitations of scaling to dozens or more patients. Induced neuronal (iN) cells reprogrammed directly from dermal fibroblasts or urinary epithelia could be obtained from many donors, but such donor cells are heterogeneous, show interindividual variability, and must be extensively expanded, which can introduce random mutations. Moreover, derivation of dermal fibroblasts requires invasive biopsies. Here we show that human adult peripheral blood mononuclear cells, as well as defined purified T lymphocytes, can be directly converted into fully functional iN cells, demonstrating that terminally differentiated human cells can be efficiently transdifferentiated into a distantly related lineage. T cell-derived iN cells, generated by nonintegrating gene delivery, showed stereotypical neuronal morphologies and expressed multiple pan-neuronal markers, fired action potentials, and were able to form functional synapses. These cells were stable in the absence of exogenous reprogramming factors. Small molecule addition and optimized culture systems have yielded conversion efficiencies of up to 6.2%, resulting in the generation of >50,000 iN cells from 1 mL of peripheral blood in a single step without the need for initial expansion. Thus, our method allows the generation of sufficient neurons for experimental interrogation from a defined, homogeneous, and readily accessible donor cell population.

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