Separation of functionally divergent muscle precursor cell populations from porcine juvenile muscles by discontinuous Percoll density gradient centrifugation.

BACKGROUND: Satellite cells (SC) and their descendants, muscle precursor cells (MPC), play a key role in postnatal muscle development, regeneration, and plasticity. Several studies have provided evidence that SC and MPC represent a heterogeneous population differing in their biochemical and functional properties. The identification and characterization of functionally divergent SC subpopulations should help to reveal the precise involvement of SC/MPC in these myogenic processes. The aim of the present work was therefore to separate SC subpopulations by using Percoll gradients and to characterize their myogenic marker profiles and their functional properties (adhesion, proliferation, and differentiation). RESULTS: SC/MPC from muscles of 4-day-old piglets were isolated by trypsin digestion and enriched by Percoll density gradient centrifugation. A mixed myogenic cell population was obtained from the 40/70% interface (termed: mixed P40/70) of a 25/40/70% Percoll gradient. Thereafter, by using a more stepped 25/40/50/70% Percoll gradient, mixed P40/70 was divided into subpopulation 40/50 (SP40/50) collected from the 40/50% interface and subpopulation 50/70 (SP50/70) collected from the 50/70% interface. All three isolated populations proliferated and showed a myogenic phenotype characterized by the ability to express myogenic markers (Pax7, MyoD1, Desmin, and MyoG) and to differentiate into myotubes. However, compared with mixed P40/70, SP40/50 and SP50/70 exhibited distinct functional behavior. Growth kinetic curves over 90 h obtained by the xCELLigence system and proliferation assays revealed that SP40/50 and mixed P40/70 constituted a fast adhering and fast proliferating phenotype. In contrast, SP50/70 showed considerably slower adhesion and proliferation. The fast-proliferating SP40/50 showed the highest myogenic differentiation potential with higher fusion rates and the formation of more middle-sized and large myotubes. CONCLUSIONS: The described Percoll density gradient centrifugation represents a useful tool for subdividing pig SC/MPC populations with divergent myogenic functions. The physiological role of SC subpopulations during myogenesis and the interaction of these populations can now be analyzed to a greater extent, shedding light on postnatal growth variations in pigs and probably in other animals.

A hypomorphic BMPR1B mutation causes du Pan acromesomelic dysplasia.

BACKGROUND: Grebe dysplasia, Hunter-Thompson dysplasia, and du Pan dysplasia constitute a spectrum of skeletal dysplasias inherited as an autosomal recessive trait characterized by short stature, severe acromesomelic shortening of the limbs, and normal axial skeleton. The majority of patients with these disorders have biallelic loss-of-function mutations of GDF5. In single instances, Grebe dysplasia and a Grebe dysplasia-like phenotype with genital anomalies have been shown to be caused by mutations in BMPR1B, encoding a GDF5 receptor. METHODS: We clinically and radiologically characterised an acromesomelic chondrodysplasia in an adult woman born to consanguineous parents. We sequenced GDF5 and BMPR1B on DNA of the proposita. We performed 3D structural analysis and luciferase reporter assays to functionally investigate the identified BMPR1B mutation. RESULTS: We extend the genotype-phenotype correlation in the acromesomelic chondrodysplasias by showing that the milder du Pan dysplasia can be caused by a hypomorphic BMPR1B mutation. We show that the homozygous c.91C>T, p.(Arg31Cys) mutation causing du Pan dysplasia leads to a significant loss of BMPR1B function, but to a lesser extent than the previously reported p.Cys53Arg mutation that results in the more severe Grebe dysplasia. CONCLUSIONS: The phenotypic severity gradient of the clinically and radiologically related acromesomelic chondrodysplasia spectrum of skeletal disorders may be due to the extent of functional impairment of the ligand-receptor pair GDF5-BMPR1B.