Induced pluripotent stem cell reprogramming by integration-free Sendai virus vectors from peripheral blood of patients with craniometaphyseal dysplasia.
Studies of rare genetic bone disorders are often limited due to unavailability of tissue specimens and the lack of animal models fully replicating phenotypic features. Craniometaphyseal dysplasia (CMD) is a rare monogenic disorder characterized by hyperostosis of craniofacial bones concurrent with abnormal shape of long bones. Mutations for autosomal dominant CMD have been identified in the ANK gene (ANKH). Here we describe a simple and efficient method to reprogram adherent cells cultured from peripheral blood to human induced pluripotent stem cells (hiPSCs) from eight CMD patients and five healthy controls. Peripheral blood mononuclear cells (PBMCs) were separated from 5-7 mL of whole blood by Ficoll gradient, expanded in the presence of cytokines and transduced with Sendai virus (SeV) vectors encoding OCT3/4, SOX2, KLF4, and c-MYC. SeV vector, a cytoplasmic RNA vector, is lost from host cells after propagation for 10-13 passages. These hiPSCs express stem cell markers, have normal karyotypes, and are capable of forming embryoid bodies in vitro as well as teratomas in vivo. Further differentiation of these patient-specific iPSCs into osteoblasts and osteoclasts can provide a useful tool to study the effects CMD mutations on bone, and this approach can be applied for disease modeling of other rare genetic musculoskeletal disorders.
1 Department of Oral Health and Diagnostic Sciences, School of Dental Medicine, University of Connecticut Health Center , Farmington, CT, 06030.
I-Ping
IP
Chen
Keiichi
K
Fukuda
Noemi
N
Fusaki
Akihiro
A
Iida
Mamoru
M
Hasegawa
Alexander
A
Lichtler
Ernst J
EJ
Reichenberger
D000328:Adult; D001483:Base Sequence; D001848:Bone Diseases, Developmental / Q000097:blood*; D016022:Case-Control Studies; D065150:Cellular Reprogramming; D002648:Child; D019465:Craniofacial Abnormalities / Q000097:blood*; D017931:DNA Primers; D005260:Female; D015329:Gene Rearrangement, T-Lymphocyte; D005822:Genetic Vectors; D006801:Humans; D015576:Hyperostosis / Q000097:blood*; D006972:Hypertelorism / Q000097:blood*; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D029082:Sendai virus / Q000235:genetics*
D017931:DNA Primers; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4