To date, there are several well-established models of the neural crest (NC) that allow for the critical investigation of different developmental stages such as: induced pluripotent stem cells (iPSCs), NC, melanoblast and terminally differentiated melanocytes. The human iPSCs were generated in the lab following the protocol from Sommer et al. [291]. The generated human iPSCs were used in the melanocyte differentiation protocol, which was first established by Nissan and colleagues [292]. In brief, hiPSCs were seeded on mitomycin C-treated, postmitotic NIH 3T3 fibroblasts in stem cell medium. After three days the medium was switched to differentiation medium supplemented with ascorbic acid and BMP4. For a complete list of medium components see Table 2. Pigmentation was observed by day 30 (Figure 3), however only after day 60 were there large enough pigmented colonies available to be manually picked and cultured in melanocyte specific medium (Figure 3).
Upon successfully establishing the melanocyte differentiation protocol the melanocytes derived from hiPSCs, termed Mel D1, were characterized.
Common pluripotency markers were investigated to evaluate the differentiation protocol. For example, the expression of pluripotency markers OCT4 and LIN28 were downregulated with increasing time of differentiation (Figure 4a), while differentiation markers, MITF and TRP2, were upregulated with increasing time of differentiation (Figure 4b). The pigmentation of the Mel D1 cells was comparable to that of normal human melanocytes (NHMs) (Figure 4c). Additionally, the morphology of the Mel D1 was similar to that of NHMs as both melanocyte populations showed at least two dendritic processes per cells, which is a hallmark in melanocyte-specific morphology (Figure 4d). Next, expression of additional differentiation markers was investigated on protein-level using immunofluorescence. MITF, GP100 (PMEL17), TRP2 and TRP1 were detected in Mel D1 cells at levels comparable to NHMs (Figure 4e).
Mel D1 cells were further characterized using electron microscopy (EM) with the help of the DKFZ electron microscopy core facility. Mel D1 cells were seeded on special disc punches and allowed to attach and grow before they were subjected to electron microscopy (method describe in Supplemental section 8.1.2.1). Melanosome development was examined and the four stages were found in both the Mel D1 cells and NHMs (Figure 5). Melanosomes in premelanosome stage I are characterized by their spherical shape, as clearly seen in the upper panel of both the Mel D1 and NHMs (Figure 5, upper left panels). Those in premelanosome stage II are ellipsoidal in shape and contain perpendicular filaments; but have no notable pigmentation present. During stage III, the organelle starts to become partially pigmented or melanized. This partially melanized melanosome organelle was successfully identified in both Mel D1 and NHMs (Figure 5, upper middle panels). Lastly, at stage IV, the melanization process is completed and the organelle appears completely pigmented and therefore black in color [294] (Figure 5, upper right panels). These experiments provided substantial evidence that Mel D1 cells represent a population of successfully differentiated melanocytes with functionally essential features, i.e. melanosome development (Figure 4-5).
Mel D1 cells were further validated and characterized using whole genome expression analysis. Three independently generated Mel D1 populations were subjected to a whole genome gene expression microarray and global gene expression patterns were compared between hiPSCs, neural crest cells (characterization not shown, method described in Supplemental section 8.1.1.2), Mel D1 and NHMs (Figure 6a). Heatmap presentation illustrates the differentially up- and down- regulated genes determined by several groups testing with each row representing one gene and each column a sample (yellow indicates upregulation and blue downregulation).
Pearson distance shows that Mel D1 cells have strong similarities to NHMs in contrast to their derivative hiPSCs. Upon hierarchical clustering, Mel D1 and NHMs cluster closely together and exhibit similar genome-wide expression patterns (Figure 6a).
Moreover, comparable analyses were performed for Mel D1 vs. hiPSCs (group A) and NHMs vs. hiPSCs (group B). First, gene expression values for pluripotency-related markers and differentiation-related markers were extracted using Chipster software (Figure 6b). All pluripotency markers were similarly downregulated in both comparison groups, whereas differentiation markers were upregulated in both groups, shown as log2 [fold change]. Notably, expression of TRP2 detected by microarray analysis was higher in NHMs compared to expression in Mel D1, which is in contrast to expression analysis using qPCR (Figure 4b), where higher TRP2 levels were observed in Mel D1 cells. This pattern was also observed for MITF expression with higher expression levels in Mel D1 cells detected by qPCR (Figure 4b) but higher expression levels in NHMs using the gene expression microarray (Figure 6b). Ingenuity analysis (IPA) revealed that most pathways enriched in differentially expressed genes (Figure 6c, +2≤ fold change ≤-2) were similarly deregulated between Mel D1 vs. hiPSCs and NHMs vs. hiPSCs. However, a difference was observed in the pathway ‘transcriptional regulatory network in embryonic stem cells’ between the two groups with a stronger enrichment in differentially expressed genes in NHMs compared to Mel D1 cells. If this has any significant meaning remains under investigation, but it is not surprising that differentiating hiPSCs towards the melanocytic lineage would utilize slightly different canonical pathways.
More detailed analysis of the canonical pathway ‘melanocyte development and pigmentation signaling’ using IPA revealed that genes involved in the melanocyte pigmentation pathway were specifically differentially regulated when comparing Mel D1 vs. hiPSCs and NHMs vs. hiSPCs (Figure 6d). Nearly all genes that were upregulated in Mel D1 vs. hiPSCs were also upregulated when NHMs were compared to hiPSCs. In general, NHMs vs. hiPSCs pigmentation pathway was more highly upregulated compared to Mel D1 vs. hiPSCs. For example, TRP2 was slightly upregulated 2.35 and highly upregulated 6.6 fold in Mel D1 vs. hiPSCs and NHMs vs. hiPSCs respectively. Strikingly, genes essential for the melanin production in melanocytes, TRP1, TRP2 and TYR, were strongly upregulated in both NHMs and Mel D1 cells compared to hiPSCs confirming data from electron microscopy that the pigmented organelles were present in both melanocytic populations (Figure 6d).
Based on these results, I conclude that I was able to successfully differentiate hiPSCs into melanocytes. For this work, the functionality of these melanocytes was not tested, for example, in an in vitro model of reconstituted melanized epidermis or organotypic skin reconstruction. However, this should be investigated in the future.