Differentiation of Human Induced Pluripotent Stem Cells from Patients with Autosomal Recessive Congenital Ichthyosis and Analysis of Gene Expression Patterns

Autosomal recessive congenital ichthyosis (ARCI) and trichothiodystrophy (TTD) are cornification disorders of the epidermis. Both share similarities in symptoms: scaling and thickened skin, which often results in red swelling, skin fissures, and increased water loss from the epidermis. Pathomechanisms and affected genes differ between ARCI and TTD but similarities in symptoms demonstrate the complexities of epidermal formation, prompting research that provides a better understanding of the intertwining relationships between genes and proteins responsible for epidermal barrier formation.

Knockdown experiments targeting ARCI-causative genes, TGM1, ALOX12B, ALOXE3, and CERS3, were carried out utilising small interfering RNA (siRNA). Gene expression ≤5% activity was achieved for the majority of selected genes. Further analyses of genes involved in the acylceramide biosynthesis pathway were undertaken by quantified by real-time PCR (RT qPCR). Data showed a clear upregulation of all genes chosen falls in line with current research that suggests a barrier disruption elicits a compensatory effect.

Three IPSC lines, previously created from donated patient fibroblasts, were differentiated into keratinocyte progenitors, and further terminally differentiated. All three lines showed good markers for differentiation and loss of pluripotency over a 30 period, with the addition of growth factors. Markers for basal keratinocytes, keratin 14 and 5, were present, and terminal differentiation markers keratin 10, TGase-1, and filaggrin. These were imaged fluorescently and other markers quantified with RT qPCR. In particular, certain mutations these lines carry, such as the ERCC2 mutation in the TTD patient line, affected the presence of epidermal marker filaggrin, and the expected reduction in gene expression was seen in RT qPCR. The lack of filaggrin mRNA verifies the ability to use IPSC to model disease in cornification disorders of the skin.