One of the main obstacles in human studies
One of the main obstacles in human studies is sample acquisition. Human iPSC lines are usually produced from fibroblasts isolated from skin biopsies, a traumatic invasive procedure demanding local anesthesia and medical follow-up. Hair follicle (HF) plucking, however, is simple, painless and non-invasive alternative procedure. Moreover, the HF-keratinocytes, similar to neurons are derived from the same ectodermal embryonic layer, and thus, besides carrying the same genetic load, could retain the same epigenetic alterations during early development. Aasen et al. have shown that skin and HF keratinocytes could be reprogrammed into iPSC (Aasen et al., 2008; Aasen and Belmonte, 2010). In the present study we confirmed the feasibility of this approach and improved it by using 1) a polycistronic lentival vector, 2) exogenous inhibitors and 3) stored hairs. Moreover, we show that iPSC from HF (HF-iPSC) have potential to give neural progenitors and functional neurons. We propose a cellular model of iPSC obtained from few hair follicles plucked from the scalp region, utilizing it as a novel source for in vitro neural commitment to model neurodevelopmental disorders.
Results and discussion We found that as few as ten HF in anagen phase, identified by the presence of the outer root sheath, were sufficient to obtain keratinocytes for reprogramming (Fig. 1A). Notably, HF can be stored for at least 48h in plain medium at room temperature without a decrease in cell recovery, allowing shipments worldwide. Keratinocytes were detached by trypsin treatment from HF and typical keratinocyte colonies appeared 7–10days later (Fig. 1B) (Limat and Noser, 1986) for all healthy or diseased donors that we tested. Early passage keratinocytes were infected with lentiviral particles expressing the polycistronic plasmid STEMCCA (OCT4, SOX2, KLF4 and c-MYC), which allows simultaneous delivery of the 4 genes with only one integration site, maximizing reprogramming efficiency (Somers et al., 2010). A GFP control virus confirmed high infection rate of primary keratinocytes (Fig. 1C). We found that reprogramming was increased by 3.5 fold (p=0.04) with the use of SB431542, CHIR99021, Parnate and PS48 (Zhu et al., 2010). Several colonies with typical hES morphology (defined border, high nuclei/cytoplasm ratio and prominent nucleoli) were obtained and expanded individually (Fig. 1D). Pluripotency of HF-derived iPSC from 2 healthy individuals (HF-iPSC-1 and HF-iPSC-2) was confirmed by the AP1903 of the pluripotency markers Pou5F1 (Oct3/4), SOX2, NANOG and DNMT3B (Fig. 2A) as well as by TRA-1-81, SSEA-4 and alkaline phosphatase (Fig. 2B). Of note, transgene expression was found to be efficiently silenced (Suppl. Fig. 1A). HF-iPSC lines were analyzed in the PluriTest assay, a novel bioinformatic assay for pluripotency, which eliminates the need of animal use for teratoma assay (Müller et al., 2011). Both HF-iPSC lines displayed high Pluripotency Scores, similar to hES and fibroblast-derived iPSC (Fig. 2C). HF-iPSC lines scored relatively high on PluriTest\'s Novelty Score, a metric that measures the divergence of the global stem cell transcriptome from expected patterns present in well-characterized iPSC and hES. This result suggests that HF-iPSC, while being genetically normal as demonstrated by karyotypic analysis (Suppl. Fig. 1B), might display a distinct signature, possibly due ‘epigenetic memory’ stemming from their ectodermal origin. HF-iPSC pluripotency was further demonstrated by their in vitro ability to form embryoid bodies (EB) and to differentiate into the three embryonic germ layers endoderm (shown by the expression of alpha-fetoprotein (AFP)), mesoderm (CD31) and ectoderm (cytokeratin K14) (Fig. 2D). Spontaneous contraction was observed indicating cardiac commitment (data not shown), which was further confirmed by Troponin T (cardiac marker) positive cells (Fig. 2D). Characterized pluripotent HF-iPSC were further differentiated into neuronal cells. During the last decade, studies on hES have reported numerous protocols for neuronal differentiation, leading to the production of homogenous and specialized neurons, capable of basic neuronal activities in vitro and upon transplantation in animal models. We evaluated the neural potential of HF-iPSC to form two types of multipotent progenitors, either anterior neuroectodermal Pax6+ cells or floor plate FoxA2+ cells. The two HF-iPSC lines spontaneously formed rosette structures that contained Pax6+/nestin+ neural progenitor cells upon culturing them on MS-5 stroma cells in absence of morphogens (Fig. 3A-a). Moreover, HF-iPSC were also differentiated into floor plate FOXA2+ progenitors, a newly described type of neural precursors (Fasano et al., 2010) (Fig. 3A-b). Quantification by flow cytometry showed that 89% of the cells were FOXA2+ (Fig. 3A-c). In order to fully evaluate the neuronal potential of HF-iPSC, we directed the HF-iPSC to two types of mature neurons, dopaminergic neurons or forebrain neurons, which are involved in various neurological disorders. HF-iPSC were differentiated towards mature βIII-tubulin+ TH+ dopaminergic neurons by SMAD inhibition protocol (Chambers et al., 2009) (Fig. 3B-a). Quantification by flow cytometry showed that 75% of the cells are βIII-tubulin+ (Fig.3B-b). HPLC analysis of the extracellular medium of these differentiated neural cells revealed the presence of both dopamine and serotonin, and their respective metabolites homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5HIAA) (Fig. 3B-c), while they were undetected in undifferentiated HF-iPSC (data not shown). This further supports the presence of functional dopaminergic and serotonergic neural cells in our differentiated culture. In parallel, HF-iPSC could also be differentiated towards βIII-tubulin+/TBR1+ forebrain glutamatergic neurons by prolonged spontaneous EB differentiation (Fig. 3C).