Publications by authors named "Ekaterina P Kalabusheva"

6 Publications

  • Page 1 of 1

Generation of Hair Follicle Germs In Vitro Using Human Postnatal Skin Cells.

Methods Mol Biol 2020 ;2154:153-163

Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Moscow, Russia.

Modeling organoids with hair follicle germ-like properties provides an opportunity for developing strategies for alopecia drug discovery and replacement therapy, as well as investigating the molecular mechanisms underlying human hair follicle regeneration in vitro. Hair follicle germ reconstruction in vitro is based on dermal papilla hair-inducing abilities and the plasticity of skin epidermal keratinocytes. The current protocol describes a highly efficient approach suitable for adult human skin cell applications. This method allows to obtain hair follicle germs using tissues from one donor. Isolated and cultured for 2 weeks, adult hair follicle dermal papilla cells and skin epidermal keratinocytes self-organize in hanging drop cultures generating organoids that exhibit the features of folliculogenesis onset.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-0648-3_13DOI Listing
March 2021

Metabolic activity and intracellular pH in induced pluripotent stem cells differentiating in dermal and epidermal directions.

Methods Appl Fluoresc 2019 Sep 9;7(4):044002. Epub 2019 Sep 9.

Privolzhsky Research Medical University, 10/1 Minin and Pozharsky sq., Nizhny Novgorod, 603950, Russia. Lobachevsky State University of Nizhny Novgorod, 23 Gagarin Avenue, Novgorod, Nizhny Novgorod, 603950, Russia.

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1088/2050-6120/ab3b3dDOI Listing
September 2019

C-TALE, a new cost-effective method for targeted enrichment of Hi-C/3C-seq libraries.

Methods 2020 01 26;170:48-60. Epub 2019 Jun 26.

Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia. Electronic address:

Studies performed using Hi-C and other high-throughput whole-genome C-methods have demonstrated that 3D organization of eukaryotic genomes is functionally relevant. Unfortunately, ultra-deep sequencing of Hi-C libraries necessary to detect loop structures in large vertebrate genomes remains rather expensive. However, many studies are in fact aimed at determining the fine-scale 3D structure of comparatively small genomic regions up to several Mb in length. Such studies typically focus on the spatial structure of domains of coregulated genes, molecular mechanisms of loop formation, and interrogation of functional significance of GWAS-revealed polymorphisms. Therefore, a handful of molecular techniques based on Hi-C have been developed to address such issues. These techniques commonly rely on in-solution hybridization of Hi-C/3C-seq libraries with pools of biotinylated baits covering the region of interest, followed by deep sequencing of the enriched library. Here, we describe a new protocol of this kind, C-TALE (Chromatin TArget Ligation Enrichment). Preparation of hybridization probes from bacterial artificial chromosomes and an additional round of enrichment make C-TALE a cost-effective alternative to existing many-versus-all C-methods.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymeth.2019.06.022DOI Listing
January 2020

Regeneration of Dermis: Scarring and Cells Involved.

Cells 2019 06 18;8(6). Epub 2019 Jun 18.

Laboratory of Cell Biology, N.K. Koltsov Institute of Developmental Biology, 26 Vavilov str., Moscow 119334, Russia.

There are many studies on certain skin cell specifications and their contribution to wound healing. In this review, we provide an overview of dermal cell heterogeneity and their participation in skin repair, scar formation, and in the composition of skin substitutes. The papillary, reticular, and hair follicle associated fibroblasts differ not only topographically, but also functionally. Human skin has a number of particular characteristics that are different from murine skin. This should be taken into account in experimental procedures. Dermal cells react differently to skin wounding, remodel the extracellular matrix in their own manner, and convert to myofibroblasts to different extents. Recent studies indicate a special role of papillary fibroblasts in the favorable outcome of wound healing and epithelial-mesenchyme interactions. Neofolliculogenesis can substantially reduce scarring. The role of hair follicle mesenchyme cells in skin repair and possible therapeutic applications is discussed. Participation of dermal cell types in wound healing is described, with the addition of possible mechanisms underlying different outcomes in embryonic and adult tissues in the context of cell population characteristics and extracellular matrix composition and properties. Dermal white adipose tissue involvement in wound healing is also overviewed. Characteristics of myofibroblasts and their activity in scar formation is extensively discussed. Cellular mechanisms of scarring and possible ways for its prevention are highlighted. Data on keloid cells are provided with emphasis on their specific characteristics. We also discuss the contribution of tissue tension to the scar formation as well as the criteria and effectiveness of skin substitutes in skin reconstruction. Special attention is given to the properties of skin substitutes in terms of cell composition and the ability to prevent scarring.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells8060607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627856PMC
June 2019

A mosaic intragenic microduplication of LAMA1 and a constitutional 18p11.32 microduplication in a patient with keratosis pilaris and intellectual disability.

Am J Med Genet A 2018 11 23;176(11):2395-2403. Epub 2018 Sep 23.

Laboratory of Cytogenetics, Research Institute of Medical Genetics, Tomsk NRMC, Tomsk, Russia.

The application of array-based comparative genomic hybridization and next-generation sequencing has identified many chromosomal microdeletions and microduplications in patients with different pathological phenotypes. Different copy number variations are described within the short arm of chromosome 18 in patients with skin diseases. In particular, full or partial monosomy 18p has also been associated with keratosis pilaris. Here, for the first time, we report a young male patient with intellectual disability, diabetes mellitus (type I), and keratosis pilaris, who exhibited a de novo 45-kb microduplication of exons 4-22 of LAMA1, located at 18p11.31, and a 432-kb 18p11.32 microduplication of paternal origin containing the genes METTL4, NDC80, and CBX3P2 and exons 1-15 of the SMCHD1 gene. The microduplication of LAMA1 was identified in skin fibroblasts but not in lymphocytes, whereas the larger microduplication was present in both tissues. We propose LAMA1 as a novel candidate gene for keratosis pilaris. Although inherited from a healthy father, the 18p11.32 microduplication, which included relevant genes, could also contribute to phenotype manifestation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajmg.a.40478DOI Listing
November 2018

Multimodal label-free imaging of living dermal equivalents including dermal papilla cells.

Stem Cell Res Ther 2018 04 3;9(1):84. Epub 2018 Apr 3.

Institute of Biomedical Technologies, Nizhny Novgorod State Medical Academy, Minin and Pozharsky Square, 10/1, Nizhny Novgorod, 603005, Russia.

Background: Despite the significant progress in the development of skin equivalents (SEs), the problem of noninvasively assessing the quality of the cell components and the collagen structure of living SEs both before and after transplantation remains. Undoubted preference is given to in vivo methods of noninvasive, label-free monitoring of the state of the SEs. Optical bioimaging methods, such as cross-polarization optical coherence tomography (CP OCT), multiphoton tomography (MPT), and fluorescence lifetime imaging microscopy (FLIM), present particular advantages for the visualization of such SEs.

Methods: In this study, we simultaneously applied several visualization techniques for skin model examination. We investigated the structure and quality of dermal equivalents containing dermal papilla (DP) cells and dermal fibroblasts (FBs) using CP OCT, MPT, and FLIM. Both the energy metabolism of the cell components and the structuring of the collagen fibrils were addressed.

Results: Based on the data from the fluorescence lifetimes and the contributions of protein-bound NAD(P)H, a bias toward oxidative metabolism was indicated, for the first time, in both the DP cells and FBs on day 14 of SE cultivation. The CP OCT and MPT data also indicated that both DP cells and FBs structured the collagen gel in a similar manner.

Conclusion: In this study, multimodal label-free imaging of the structure and quality of living dermal equivalents was implemented for the first time with the use CP OCT, MPT, and FLIM of NAD(P)H. Our data suggest that the combination of different imaging techniques provides an integrated approach to data acquisition regarding the structure and quality of dermal equivalents, minimizes the potential disadvantages of using a single method, and provides an ideal information profile for clinical and research applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13287-018-0838-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5883517PMC
April 2018