CDK4 and cyclin D1 allow human myogenic cells to recapture growth property without compromising differentiation potential

Abstract

In vitro culture systems of human myogenic cells contribute greatly to elucidation of the molecular mechanisms underlying terminal myogenic differentiation and symptoms of neuromuscular diseases. However, human myogenic cells have limited ability to proliferate in culture. We have established an improved immortalization protocol for human myogenic cells derived from healthy and diseased muscles; constitutive expression of mutated cyclin-dependent kinase 4, cyclin D1 and telomerase immortalized human myogenic cells. Normal diploid chromosomes were preserved after immortalization. The immortalized human myogenic cells divided as rapidly as primary human myogenic cells during the early passages, and underwent myogenic, osteogenic and adipogenic differentiation under appropriate culture conditions. The immortalized cells contributed to muscle differentiation upon xenotransplantation to immunodeficient mice under conditions of regeneration following muscle injury. We also succeeded in immortalizing cryopreserved human myogenic cells derived from Leigh disease patients following primary culture. Forced expression of the three genes shortened their cell cycle to o30 h, which is similar to the doubling time of primary cultured human myogenic cells during early passages. The immortalization protocol described here allowed human myogenic cells to recapture high proliferation activity without compromising their differentiation potential and normal diploidy.

Lipin1 Regulates Skeletal Muscle Differentiation through Extracellular Signal-regulated Kinase (ERK) Activation and Cyclin D Complex-regulated Cell Cycle Withdrawal

“…In contrast, cyclin D3 overexpression promotes myogenic differentiation as demonstrated by increased MyoD and MyoG expression (37). Unlike cyclin D3, cyclin D1⅐CDK4 activity is focused on myoblast proliferation and is reported to suppress skeletal muscle differentiation by blocking the activity of the MEF2 family of transcriptional regulators (39,40).…”

Establishment of a Human Nonluteinized Granulosa Cell Line that Transitions from the Gonadotropin-Independent to the Gonadotropin-Dependent Status

“…Recently, CDK4R24C and cyclin D1, which phosphorylate pRB so as to inactivate it, and hTERT have been used to immortalize human ovary surface epithelial cells, myoblasts, and tongue and dermal keratinocytes without inducing genomic abnormality (21,28,29). Based on the results, we first transduced CDK4R24C, cyclin D1, and hTERT into primary non-LGC, but it was not sufficient to immortalize them.…”

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators

“…Since the genome and cellular condition of K4DT cells would be intact (Shiomi et al 2011), immortalized K4DT cell of Lowland Anoa would be useful for genome, transcriptome, and proteome analyses, which would provide valuable information about the evolution and divergence of Bubalus and related species, at the molecular level (Kikkawa et al 1997). To our knowledge, this study is the first report about the efficient establishment of immortalized cells from endangered animals.…”

“…Our group reported earlier that expression of mutant CDK4 (R24C), Cyclin D, and TERT allows us to induce the infinite cell proliferation (immortalization) of the bovine and porcine derived cells. These cells are named K4DT (CDK4-Cyclin D-TERT) cells (Donai et al 2014;Kuroda et al 2015a, b;Shiomi et al 2011) from their name of the introduced genes. Since the K4DT cell lines infinitely proliferate with keeping the original nature of the cells (Shiomi et al 2011).…”

“…These cells are named K4DT (CDK4-Cyclin D-TERT) cells (Donai et al 2014;Kuroda et al 2015a, b;Shiomi et al 2011) from their name of the introduced genes. Since the K4DT cell lines infinitely proliferate with keeping the original nature of the cells (Shiomi et al 2011). In brief, the immortalized myoblasts keep the ability to differentiate into myogenic, osteogenic, and adipogenic cells.…”