Cell Engineering and Gene Editing Approaches Towards Correcting Human Skin Pigmentation Disorders | AIChE

Cell Engineering and Gene Editing Approaches Towards Correcting Human Skin Pigmentation Disorders


Conference Presentation

Conference Type

AIChE Annual Meeting

Presentation Date

November 8, 2021


18 minutes

Skill Level




Melanin is a general term used for heterogenous and structurally ill-defined biopolymer 1 that encompasses two forms of melanin namely black/brown eumelanin and red/yellow pheomelanin. Melanocytes (MC), which are developmentally derived from a transient embryonic cell population termed as neural crest stem cells2,3, synthesize melanin within the specialized organelles called melanosomes4, which upon maturation are transported to the surrounding epidermal keratinocytes (KC) resulting in skin pigmentation. Skin hypopigmentation diseases such as vitiligo, where amelanotic skin patches affects quality of life of the patients leading to psychological problems such as stress, embarrassment, anxiety, and social stigma, affects ~1% world’s population5. Current clinical treatment of vitiligo relies on UV radiation treatment of skin in a hope to activate residual MC precursor cells within the hair follicle infundibulum and bulge to differentiate into mature melanin producing MC and repopulate basal epidermis. However, this approach results poor clinical outcome due to inefficient and inadequate repigmentation of skin. Alternatively, transplanting autologous tissue engineered skin grafts, made of MC and KC derived from patient’s induced pluripotent stem cells (iPSC), is an attractive strategy. However, controlling precise amount of melanin synthesis from MC of the engineered skin in order to match color and tone of patient’s skin remains a challenge. The lack of complete understanding of the genes/pathways involved in MC precursor’s maturation into melanin producing MC presents itself as a major barrier towards developing effective skin repigmentation therapies.

In the present work, we attempted to fill this knowledge gap by performing a genetic screen to identify genes directly involved in melanosome maturation. By exploiting light scattering properties of melanin (measured as side scatter (SSC) parameter of flow cytometer), we first developed an assay to measure melanin levels of live pigment cells and then examined the extent to which changes in cellular melanin content influence SSC and whether SSC can be used to capture dynamic changes in the melanin levels upon inducing genetic perturbations. Here, we demonstrate that cellular melanin concentration indeed determines light scattering (SSC) properties of pigments cells and SSC can be used as a proxy for melanin levels to perform a CRISPR-Cas9 based genetic screen to identify novel regulators of human melanogenesis. In order to establish a link between melanin content and SSC, we modeled human melanogenesis in vitro using pluripotent stem cells in a step-wise manner, where we first obtained neural crest (NC) stem cells (SOX10+PAX3+), followed by MITF+KIT+ unpigmented MC precursors called melanoblasts (MB) and finally TYR+DCT+, melanosome-containing mature pigmented MC. We measured the total melanin content and SSC of MB as they matured into MC and noted that increase in melanin content, as measured by optical density (OD) of cell lysates at 400nM, was accompanied by increase in SSC (R2 = 0.82). We reasoned that close correlation between melanin content and SSC could be used as a paradigm for a CRISPR-Cas9 genetic screen, in which loss of genes important for melanin synthesis within melanosome would result in diminished SSC. We engineered MNT1 pigmented melanoma cells to express spCas9 nuclease upon doxycycline treatment and infected it with a genome-wide lentiviral sgRNA library (with 10 targeting-guides per gene) 6, such that each cell expressed a single sgRNA. Following puromycin selection to kill noninfected cells, expression of Cas9 was induced to carry out gene-editing, and after two weeks, top 10% low and high SSC cells were FACS sorted. As expected, low SSC fraction was enriched for hypo-pigmented cells. Genomic DNA was subsequently isolated and the frequencies of sgRNAs in both populations (low and high SSC) were measured by deep sequencing and analyzed using Cas9 high-Throughput maximum Likelihood Estimator (CasTLE) algorithm, which identified 169 putative regulators of melanogenesis at 10% false discovery rate (FDR). Gene ontology analysis of screen hits revealed associations with melanosome organization, pigment granule organization, vesicle organization and intracellular transport. We further validated select screen hits through secondary CRISPR-cas9 knockout experiments and show that they regulate distinct steps of melanosome maturation by transmission electron microscopy (TEM). As our screen identified genes, which promoted melanin synthesis, we reasoned that screen hit genes should be differentially expressed in dark skin MC compared to light skin MC in accordance to their melanin promoting role. Indeed, the RNA-seq experiments (n=30, MC derived from diversely pigmented human donors) showed that majority of screen hits were highly expressed in darkly pigmented MC further confirming their role as melanogenesis activators.

Together, these results greatly expand the known repertoire of genes involved in human melanogenesis and point towards new pathways that control pigmentation in health and disease. This study will catalyze drug discovery and skin tissue engineering based therapies against vitiligo and other hypopigmentation disorders.

1 Meredith, P. & Sarna, T. The physical and chemical properties of eumelanin. Pigment Cell Res 19, 572-594, doi:10.1111/j.1600-0749.2006.00345.x (2006).

2 Sturm, R. A. Molecular genetics of human pigmentation diversity. Hum Mol Genet 18, R9-17, doi:10.1093/hmg/ddp003 (2009).

3 Yamaguchi, Y., Brenner, M. & Hearing, V. J. The regulation of skin pigmentation. The Journal of biological chemistry 282, 27557-27561, doi:10.1074/jbc.R700026200 (2007).

4 Raposo, G. & Marks, M. S. Melanosomes--dark organelles enlighten endosomal membrane transport. Nature reviews. Molecular cell biology 8, 786-797, doi:10.1038/nrm2258 (2007).

5 Ezzedine, K., Eleftheriadou, V., Whitton, M. & van Geel, N. Vitiligo. Lancet 386, 74-84, doi:10.1016/S0140-6736(14)60763-7 (2015).

6 Morgens, D. W. et al. Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens. Nature communications 8, 15178, doi:10.1038/ncomms15178 (2017).


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