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Mechanistic Analysis Of Neurofacial Development Using Patient-Specific Stem Cells Identifies Key Genetic Determinants
Janina Kueper, MD1, Casey Tsimbal, BS1, Brahm Agnihotri1, Nikita Myers, BS1, Nikkola Carmichael, MSc2, Victoria Perroni, MBA2, Richard L. Maas, MD, PhD2, Eric C. Liao, MD, PhD1.
1Center for Regenerative Medicine, Shriners Hospital for Children/Massachusetts General Hospital, Boston, MA, USA, 2Brigham and Women's Hospital, Boston, MA, USA.



PURPOSE:
Craniofacial development is highly complex and relies on the orchestration of gene regulatory networks. During embryogenesis, the face and cortical tissue develop with close physical, cellular and molecular association. The growth and migration of juxtaposed cell populations come to form morphologically and functionally distinct parts of the neurocranial skeleton. In order to gain mechanistic insight into the associated development of cranial and cortical tissues, we examined the genetic pathogenesis of a subject who presented with combined congenital craniofacial and cerebral anomalies.
METHODS:
Whole genome sequencing was performed on a child affected by a complex syndromic cleft combined with a brain malformation and her unaffected father to identify candidate pathogenic variants. Patient and father blood samples were then used to generate induced Pluripotent Stem Cells (iPSC). The cells were derived using viral induction of the pluripotency factors OCT-4, KLF-4, c-MYC, and SOX2 in peripheral blood mononuclear cells. Cellular reprogramming was confirmed and embryoid bodies were derived from the iPSC to molecularly characterize the expression of key craniofacial, eye, and brain development genes. The iPSC were then directly differentiated in to cells relevant to neurofacial development. The in vitro molecular and cellular phenotypes were also assayed in the zebrafish, using gene expression and disruption methods.
RESULTS:
A pediatric subject presented with oblique facial cleft, anophthalmia and contralateral coloboma, as well as partial bilateral coronal synostosis, ventriculomegaly and an unusually rotated corpus callosum. The derived iPSC displayed properties typical of embryonic stem cells, such as persistent self-renewal and the expression of markers of pluripotency. All iPSC proved capable of differentiation into all germ layers. Furthermore, all iPSC proved capable of forming neural crest cells. The capacity of multi-lineage differentiation of the mutant iPSCs was maintained, with all cells beings able to differentiate into chondrocytes, adipocytes, Schwann cells, and osteocytes. Whole Genome Sequencing and in silico analysis of the subject and her father identified TDGF1 and TLE2 as potential candidate genes. Both have been previously broadly implicated in the development of the head. Gene disruption of TDGF1 gene in the zebrafish further substantiated our findings that craniofacial and cortical development overlap developmentally, disruption of which may result in combined neurofacial phenotypes.
CONCLUSION:
Using gene sequencing and editing technology in iPSC and zebrafish models, we are increasingly able to dissect the molecular and cellular basis of complex craniofacial malformations. This study illustrates application of patient-derived stem cell and animal models to gain mechanistic understanding of complex human anomalies. As modern craniofacial surgeon-scientists, we can leverage our combined clinical and scientific expertise to be informed observers of biology.


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