A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin

Jiang

The American Journal of Pathology

2008

Nerve growth factor (NGF) influences the key pathological events of psoriasis: keratinocyte proliferation, angiogenesis, and T-cell activation. We have systematically examined the kinetics of NGF expression, keratinocyte proliferation, and migration of T lymphocytes in the epidermis in Koebner-induced developing psoriatic plaques. In skin traumatized by the tape-stripping method (n ‫؍‬ 12), a marked up-regulation of NGF in Koebner-positive lesions (n ‫؍‬ 7) was observed 24 hours after trauma. Synthesis of NGF reached its maximum level in the 2nd week. Furthermore, cultured keratinocytes from nonlesional skin of psoriasis patients produced 10 times higher levels of NGF compared with keratinocytes from healthy individuals. To substantiate the in vivo effect of NGF secreted by keratinocytes in psoriatic plaques, we studied psoriatic plaques and normal human skin in a SCID-human skin xenograft model. The transplanted psoriatic plaques demonstrated marked proliferation of NGF-R (p75)-positive nerve fibers compared with only a few nerves in the transplanted normal human skin. Our results demonstrate that 1) in a developing psoriatic lesion, up-regulation of NGF together with keratinocyte proliferation are early events and precede epidermotropism of T lymphocytes; 2) keratinocytes in patients with psoriasis are primed to produce elevated levels of NGF; and 3) NGF synthesized by these keratinocytes is functionally active.

Section: Discussionmentioning

confidence: 99%

Revisiting the Koebner Phenomenon

Jiang

The American Journal of Pathology

2008

Proc. Natl. Acad. Sci. U.S.A.

“…Human foreskin fibroblasts were gifts from J. Brooks (Stanford University). Other primary human fibroblasts were obtained from Coriell Cell Repositories, Camden, NJ or derived from autopsy skin samples after removal of keratinocytes and endothelial cells as described (5). The demographic information of fibroblasts is summarized in Table 1, which is published as supporting information on the PNAS web site, www.pnas.org.…”

Section: Methodsmentioning

confidence: 99%

Diversity, topographic differentiation, and positional memory in human fibroblasts

Chang

Chi

Dudoit

et al. 2002

998

832

A fundamental feature of the architecture and functional design of vertebrate animals is a stroma, composed of extracellular matrix and mesenchymal cells, which provides a structural scaffold and conduit for blood and lymphatic vessels, nerves, and leukocytes. Reciprocal interactions between mesenchymal and epithelial cells are known to play a critical role in orchestrating the development and morphogenesis of tissues and organs, but the roles played by specific stromal cells in controlling the design and function of tissues remain poorly understood. The principal cells of stromal tissue are called fibroblasts, a catch-all designation that belies their diversity. We characterized genome-wide patterns of gene expression in cultured fetal and adult human fibroblasts derived from skin at different anatomical sites. Fibroblasts from each site displayed distinct and characteristic transcriptional patterns, suggesting that fibroblasts at different locations in the body should be considered distinct differentiated cell types. Notable groups of differentially expressed genes included some implicated in extracellular matrix synthesis, lipid metabolism, and cell signaling pathways that control proliferation, cell migration, and fate determination. Several genes implicated in genetic diseases were found to be expressed in fibroblasts in an anatomic pattern that paralleled the phenotypic defects. Finally, adult fibroblasts maintained key features of HOX gene expression patterns established during embryogenesis, suggesting that HOX genes may direct topographic differentiation and underlie the detailed positional memory in fibroblasts.

“…Primary lung fibroblasts were isolated by the explants technique (Normand & Karasek, 1995) from lung specimens obtained for lung tumor resection from 14 patients with COPD and 13 subjects without clinical, morphologic, or functional signs of COPD (control subjects; Table 1). Classification of COPD severity was based on the 2003 Global Initiative for Chronic Obstructive Lung Disease criteria (Rabe et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Heme oxygenase‐1 induction attenuates senescence in chronic obstructive pulmonary disease lung fibroblasts by protecting against mitochondria dysfunction

et al. 2018

Chronic obstructive pulmonary disease (COPD) is associated with lung fibroblast senescence, a process characterized by an irreversible proliferation arrest associated with secretion of inflammatory mediators. ROS production, known to induce senescence, is increased in COPD fibroblasts and mitochondria dysfunction participates in this process. Among the battery of cellular responses against oxidative stress damage, heme oxygenase (HO)‐1 plays a critical role in defending the lung against oxidative stress and inflammation. Therefore, we investigated whether pharmacological induction of HO‐1 by chronic hemin treatment attenuates senescence and improves dysfunctional mitochondria in COPD fibroblasts. Fibroblasts from smoker controls (S‐C) and COPD patients were isolated from lung biopsies. Fibroblasts were long‐term cultured in the presence or absence of hemin, and/or ZnPP or QC‐15 (HO‐1 inhibitors). Lung fibroblasts from smokers and COPD patients displayed in long‐term culture a senescent phenotype, characterized by a reduced replicative capacity, an increased senescence and inflammatory profile. These parameters were significantly higher in senescent COPD fibroblasts which also exhibited decreased mitochondrial activity (respiration, glycolysis, and ATP levels) which led to an increased production of ROS, and mitochondria biogenesis and impaired mitophagy process. Exposure to hemin increased the gene and protein expression level of HO‐1 in fibroblasts and diminished ROS levels, senescence, the inflammatory profile and simultaneously rescued mitochondria dysfunction by restoring mitophagy in COPD cells. The effects of hemin were abolished by a cotreatment with ZnPP or QC‐15. We conclude that HO‐1 attenuates senescence in COPD fibroblasts by protecting, at least in part, against mitochondria dysfunction and restoring mitophagy.