Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Summary

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Abstract

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

Introduction

Human pluripotent stem cells (hPSCs) provide a platform to mimic normal differentiation in a scalable fashion for disease modeling, drug screening, and cell replacement therapies ^1-6. Of particular interest, hPSCs open up avenues for studying difficult to isolate or rare/transient cell types where patient samples are scarce. Furthermore, induced pluripotent stem cells (iPSCs) enable researchers to study development and disease modeling in a patient specific manner to unravel unique mechanisms ^1,2,7-11. The previously published protocol for differentiation of melanocytes from hPSCs requires up to 6 weeks of differentiations and involves culturing ....

Protocol

NOTE: The melanocyte protocol outlined here was first demonstrated by Mica et al.

1. Preparation of Culture Medium, Coated Dishes and Maintenance of hPSCs

1. Medium Preparation
   Note: Store all medium at 4 °C in the dark for up to 2 weeks. Filter all medium for sterilization.
   1. Prepare DMEM/10% FBS. Mix 885 ml DMEM, 100 ml FBS, 10 ml Pen/Strep and 5 ml L-Glutamine. Filter for sterilization.
   2. Prepare hESC-medium. Mix 800 ml DMEM/F12, 200 ml KSR, 5 ml L-Glutamine, 10 ml MEM minimum essential amino acids solution, 1 ml β-Mercaptoethanol, and 5 ml Pen/Strep. After filtering add 10 ng/ml FGF-2.
   ....

Results

This protocol provides a method for deriving fully pigmented, mature melanocytes from hPSCs in an in vitro feeder-free, cost efficient, and reproducible manner. In contrast to the previously established Fang et al. protocol for hPSC-derived melanocytes, the outlined protocol does not require conditioned medium and decreases the time requirement. The Fang et al. protocol utilized conditioned medium from a WNT3A-producing murine cell line and took up to 6 weeks to.......

Discussion

For the successful differentiation of melanocytes from hPSCs the following suggestions should be taken into consideration. First and foremost, it is essential to work under sterile culture conditions at all times. Additionally, it is important to start with pluripotent, fully undifferentiated hPSCs; if the starting population contains differentiated cells the yield will invariably drop as the contaminants cannot be directed towards melanocytes and may even further disrupt the properly differentiating cells.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accutase	Innovative Cell Technologies	AT104
apo human transferrin	Sigma	T1147
Ascorbic Acid (L-AA)	Sigma	A4034	100 mM
B27 (B27 Supplement)	Invitrogen	17504044
β-Mercaptoethanol	Gibco-Life Technologies	21985-023	10 mg/ml
BMP4	R&D Systems	314-bp
CHIR99021	Tocris-R&D Systems	4423	6 mM
Cholera toxin	Sigma	C8052	50 mg/ml
cAMP (cyclicAMP)	Sigma	D0627	100 mM
Dexamethasone	Sigma	D2915-100MG	50 μM
DMEM - Dulbecco's Modified Eagle Medium	Gibco-Life Technologies	11985-092
DMEM/F12 - Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12	Gibco-Life Technologies	1133--032
DMEM/F12 powder	Invitrogen	12500-096
EDN3 (Endothelin-3, human)	American Peptide Company	88-5-10B	100 μM
Fibronectin	BD Biosciences	356008	200 μg/ml
gelatin (PBS without Mg/Ca)	in house		0.1% in PBS
Glucose	Sigma	G7021
Human insulin	Sigma	I2643
ITS+ Universal Culture Supplement Premix	BD Biosciences	354352
KSR (Knockout Serum Replacement)	Gibco-Life Technologies	10828-028
Knockout DMEM	Gibco-Life Technologies	10829-018
L-Glutamine	Gibco-Life Technologies	25030-081
LDN193189	Stemgent	04-0074	100 mM
Low glucose DMEM	Invitrogen	11885-084
Matrigel matrix	BD Biosciences	354234	Dissolve 1:20 in DMEM/F12
MCDB201 Medium	Sigma	M6770
MEM minimum essential amino acids solution	Gibco-Life Technologies	11140-080
Mouse embryonic fibroblasts (7 million cells/vial)	GlobalStem	GSC-8105M
Mouse Laminin-I	R&D Systems	3400-010-01	1 mg/ml
Neurobasal medium	Invitrogen	21103049
Penicilin/Streptomycin	Gibco-Life Technologies	15140-122	10,000 U/ml
Poly-L Omithin hydrobromide	Sigma	P3655	15 mg/ml
Progesterone	Sigma	P8783	0.032 g in 100 ml 100% ethanol
Putrescine dihydrochloride	Sigma	P5780
FGF2 (Recombinant human FGF basic)	R&D Systems	233-FB-001MG/CF	10 mg/ml
SB431542	Tocris-R&D Systems	1814	10 mM
Selenite	Sigma	S5261
Sodium Bicarbonate	Sigma	S5761
SCF (Stem Cell Factor, recombinant Human)	Peprotech Inc.	300-07	50 μg/ml
TYRP1 (G-17) Antibody	Santa Cruz	10443	1:200
TYRP2 Antibody	Abcam	74073	1:200
Trypsin-EDTA (0.05%)	Gibco-Life Technologies	25300-054
Y-27632 dihydrochloride	Tocris-R&D Systems	1254	10 mM