טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentNava Salman-Kesner
SubjectDerivation and Characterization of Induced Pluripotent
Stem Cells (iPSC) from Hair Follicles: a Model
for Neurodevelopmental Disorders
DepartmentDepartment of Medicine
Supervisors Full Professor Aberdam Daniel
Full Professor Ben-Shachar Dorit
Full Thesis textFull thesis text - English Version


Abstract

Reprogramming of somatic cells into embryonic-like pluripotent cells is a novel technology that enables studying cell development and differentiation. The potential of induced pluripotent stem cells (iPSCs) as unique research tool appears to be even greater in complex neuropsychiatric disorders linked to abnormal neurodevelopment, such as schizophrenia.

We hypothesized that keratinocytes isolated from hair follicles (HF) are an ideal source of patients' cells for reprogramming, due to their accessibility and their common neuroectodermal origin with neurons, which can be important for potential epigenetic memory.

We established culture conditions to isolate and expand keratinocytes from HF. Keratinocytes were infected with a lentivirus carrying a poly-cistronic cassette expressing OCT3/4, SOX2, KLF4 and cMYC. iPSC colonies were obtained and amplified from two control individuals and one patient. Pluripotency of iPSC was confirmed by immunofluorescence staining and mRNA expression of pluripotency markers, as well as by in-vitro differentiation into the 3 embryonic germ layers. The bioinformatics PluriTest assay showed high pluripotency score, similarly to embryonic stem cells. Moreover, we demonstrated the ability to store plucked HF in medium for at least 48h at room temperature without reducing their ability for reprogramming. Finally, we differentiated efficiently these hair follicle-derived iPSCs into neural progenitors, forebrain neurons and functional dopaminergic neurons.

 To evaluate the potential of this method for studying patient-derived iPSC related to mental disorders, SchiPSC clones were produced from one patient, and were induced to differentiate into neural progenitors and neurons. However, we discovered that the karyotype of the particular SchiPSC clone we used displayed genetic abnormalities that occurred during cell culture. Nevertheless, we observed that the SchiPSC could differentiate into forebrain glutamatergic neurons, but remained in an immature stage. Moreover, we studied the ability of the SchiPSC and WT clones to differentiate into mature dopaminergic neurons. While CTL-iPSC displayed a neuronal morphology and formed network -like structures, SchiPSC did not present morphological features of neurons, suggesting that SchiPSC have an impaired potential to differentiate into dopaminergic neurons. We could not, at this stage, conclude if the abnormal commitment of the SchiPSC was due to the abnormal karyotype or was disease-related. Additional studies need to be done with patient-derived iPSC clones with normal karyotype.

This study shows that human HF can be an attractive source for cell reprogramming and that HF-derived iPSC can be differentiated into various neural lineages, suggesting this experimental system as a promising in-vitro model to study normal and pathological neural development.