Plastic Surgery Research Council

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Requirement Of ALX1 Homeobox Transcription Factor In Craniofacial Development
Jonathan PINI, PhD, Eric C. Liao, MD, PhD.
Massachusetts General Hospital, Center for Regenerative Medicine, Harvard Medical School, Harvard Stem Cell Institute, Boston, MA, USA.

Purpose: Frontonasal dysplasia (FND), or median facial cleft syndrome, is a class of developmental abnormalities caused by incomplete growth and fusion of the central frontonasal protrusion with the paired maxillary facial prominences. Genetic aspects of FND are not well defined. Recently, mutations in the ALX gene family have been associated with FND. ALX1 is expressed in the facial mesenchyme of vertebrate embryos, in cranial neural crest cells and its cartilage derivatives. However, little is known about the function of this transcription factor during craniofacial development.
Methods: Using a human pedigree of 3 subjects in a family with heritable FND, we defined a loss-of-function ALX1 gene variant (A). Control and patient blood samples were used to generate induced Pluripotent Stem Cells (iPSCs), using Sendai virus infection. iPSCs were characterized based on their embryonic stem cells (ESCs) properties i.e. self-renewal and pluripotence. Moreover, karyotype, Sendai virus gene expression and ALX1 gene sequencing were performed to finalize the iPSCs characterization. To go further, iPSCs for both genotypes were subjected to neural crest cells differentiation to study the role of ALX1 transcription factor in craniofacial formation.
Results: Control and ALX1-/- iPSCs displayed typical embryonic stem cells properties, such as self-renewal and ESC markers expression (B). Moreover, iPSCs from both genotype display normal karyotype and don't express Sendai virus genes anymore. Finally, ALX1 coding region sequencing reveals the presence of pathogenic L165F mutation whereas Control iPSCs have a normal ALX1 gene.
The iPSC are differentiated into neural crest cells, and demonstrate phenotypes that reveal their mechanism of developmental anomaly, such as loss of function in chondrocytes differentiation.
Conclusions: Our results show that ALX1 is dispensable for iPSC reprogramming process, and initiation to form embryoid body. The use of ALX1 null iPSCs will provide a better comprehension of ALX1 biology and its essential role in craniofacial regulatory network. Importantly, we show how patient derived iPSC can be used to model craniofacial anomalies. Analysis of ALX1 function and identification of its target genes will help to better understand and innovate future treatments, through pharmacologic or CRISPR editing approaches.


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