Name: feeder-free derivation of melanocytes from human pluripotent stem cells
Uploaded: 2016-03-03T15:00:00
Description: Watch this Scientific Journal Video about Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells at JoVE.com

This work was supported by a fellowship for melanoma researchers from the Joanna M. Nicolay Foundation and by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award F31. This work was further supported through grants from NYSTEM and the Tri-institutional stem cell initiative (Starr Foundation).

NOTE: The melanocyte protocol outlined here was first demonstrated by Mica et al.
1. Preparation of Culture Medium, Coated Dishes and Maintenance of hPSCs
<ol>
	<li>Medium Preparation 
	Note: Store all medium at 4 &#176;C in the dark for up to 2 weeks. Filter all medium for sterilization.
	<ol>
		<li>Prepare DMEM/10% FBS. Mix 885 ml DMEM, 100 ml FBS, 10 ml Pen/Strep and 5 ml L-Glutamine. Filter for sterilization.</li>
		<li>Prepare hESC-medium. Mix 800 ml DMEM/F12, 200 ml KSR, 5 ml L-Gl...

<ol>
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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.
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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 cells with conditioned medium from L-Wnt3a cells 12. The protocol first presented by Mica et al. and described here produces pigmented cells in three weeks and removes the ambiguity and inconsistencies associated with conditioned medium.
Melanocytes are derived from the neural crest, a migratory population of cells unique to vertebrates. The neural crest is defined during gastrulation and represents a population of cells at the edge of the neural plate, bordering between the neural and non-neural ectoderm. During neurulation, the nervous tissue evolves from a neural plate to form neural folds, which converge at the dorsal midline resulting in the neural tube 13,14.
The neural crest cells emerge from the roof plate of the neural tube, opposite the notochord, and undergo an epithelial to mesenchymal transition before migrating away to give rise to a diverse population of differentiated cells. The fates of the crest cells are defined in part by the anatomic location of the roof plate along the body axis of the embryo. Neural crest cell derivatives include lineages characteristic of both mesoderm (smooth muscle cells, osteoblasts, adipocytes, chondrocytes) and ectoderm cells (melanocytes, Schwann cells, neurons) 14. Neural crest stem cells upregulate the transcription factor SOX10 and can be isolated by fluorescence-activated cell sorting with antibodies to p75 and HNK1.
The neural crest cells fated to become melanocytes pass through a melanoblast stage and upregulate KIT and MITF (microphthalmia-associated transcription factor) 6,21 MITF is a master regulator of melanocyte development and is a transcription factor responsible for controlling much of melanocyte development 22-24. Human melanoblasts migrate to the basal layer of the epidermis where they reside either in the hair bulge or surrounded by keratinocytes in the epidermis (forming pigmentation units) to serve as precursors to the mature, pigmented melanocytes. The differentiation and maturation of melanoblasts into pigmented melanocytes occurs concomitant with colonization of the hair bulb and expression of the melanin production pathway (TYRP1, TYR, OCA2 and PMEL) 25,26.
Isolating human melanocytes and melanoblasts from patients is expensive, difficult and limiting in quantity. This protocol enables researchers to differentiate hPSCs (induced or embryonic) into melanocytes or melanocyte precursors in a well defined, rapid, reproducible, scalable, and inexpensive method without cell sorting. The protocol was used previously to identify disease-specific defects when differentiating iPSCs from patients with pigmentation disorders .

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	<tbody>
		<tr>
			<td>Accutase</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>apo human transferrin</td>
			<td>Sigma</td>
			<td>T1147</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic Acid (L-AA)</td>
			<td>Sigma</td>
			<td>A4034</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>B27 (B27 Supplement)</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco-Life Technologies</td>
			<td>21985-023</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>314-bp</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR99021</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>4423</td>
			<td>6 mM</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>50 mg/ml</td>
		</tr>
		<tr>
			<td>cAMP (cyclicAMP)</td>
			<td>Sigma</td>
			<td>D0627</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D2915-100MG</td>
			<td>50 &#956;M</td>
		</tr>
		<tr>
			<td>DMEM - Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Gibco-Life Technologies</td>
			<td>11985-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 - Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12</td>
			<td>Gibco-Life Technologies</td>
			<td>1133--032</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 powder</td>
			<td>Invitrogen</td>
			<td>12500-096</td>
			<td></td>
		</tr>
		<tr>
			<td>EDN3 (Endothelin-3, human)</td>
			<td>American Peptide Company</td>
			<td>88-5-10B</td>
			<td>100 &#956;M</td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>BD Biosciences</td>
			<td>356008</td>
			<td>200 &#956;g/ml</td>
		</tr>
		<tr>
			<td>gelatin (PBS without Mg/Ca)</td>
			<td>in house</td>
			<td></td>
			<td>0.1% in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Human insulin</td>
			<td>Sigma</td>
			<td>I2643</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS+ Universal Culture Supplement Premix&#160;</td>
			<td>BD Biosciences</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>KSR (Knockout Serum Replacement)</td>
			<td>Gibco-Life Technologies</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout DMEM</td>
			<td>Gibco-Life Technologies</td>
			<td>10829-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Gibco-Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LDN193189</td>
			<td>Stemgent</td>
			<td>04-0074</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Invitrogen</td>
			<td>11885-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel matrix</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>Dissolve 1:20 in DMEM/F12</td>
		</tr>
		<tr>
			<td>MCDB201 Medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM minimum essential amino acids solution</td>
			<td>Gibco-Life Technologies</td>
			<td>11140-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse embryonic fibroblasts (7 million cells/vial)</td>
			<td>GlobalStem</td>
			<td>GSC-8105M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Laminin-I</td>
			<td>R&#38;D Systems</td>
			<td>3400-010-01</td>
			<td>1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin/Streptomycin</td>
			<td>Gibco-Life Technologies</td>
			<td>15140-122</td>
			<td>10,000 U/ml</td>
		</tr>
		<tr>
			<td>Poly-L Omithin hydrobromide</td>
			<td>Sigma</td>
			<td>P3655</td>
			<td>15 mg/ml&#160;</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P8783</td>
			<td>0.032g in 100ml 100% ethanol</td>
		</tr>
		<tr>
			<td>Putrescine dihydrochloride</td>
			<td>Sigma</td>
			<td>P5780</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2 (Recombinant human FGF basic)</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-001MG/CF</td>
			<td>10 mg/ml</td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1814</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>Selenite</td>
			<td>Sigma</td>
			<td>S5261</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF (Stem Cell Factor, recombinant Human)&#160;</td>
			<td>Peprotech Inc.</td>
			<td>300-07</td>
			<td>50 &#956;g/ml</td>
		</tr>
		<tr>
			<td>TYRP1 (G-17) Antibody</td>
			<td>Santa Cruz</td>
			<td>10443</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>TYRP2 Antibody</td>
			<td>Abcam</td>
			<td>74073</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%)</td>
			<td>Gibco-Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride</td>
			<td>Tocris-R&#38;D Systems</td>
			<td>1254</td>
			<td>10 mM</td>
		</tr>
	</tbody>
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feeder-free derivation of melanocytes from human pluripotent stem cells

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.

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...

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Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

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.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

The overall goal of this protocol is to differentiate human pluripotent stem cells into fully pigmented, mature melanocytes. This method can help identify key factors during melanocyte development. Additionally, this protocol will enable us to identify factors that have gone awry during disease development, as well as screen for important drugs and during drug discovery.The main advantage of this protocol is that it enables us to produce large numbers of fully pigmented melanocytes. Whereas isolating melanocytes from patients usually results in relatively few cells. After aliquoting and freezing gelatinous protein, thaw and resuspend a one milliliter aliquot in 19 milliliters of DMEM f12.Then introduce five milliliters of this mixture onto a single 10 centimeter tissue culture dish. After preparing several such plates proceed to incubate them for one hour at room temperature. Next, begin to prepare plates coated with PO, laminin, and fibronectin by first adding PBS containing 15 micrograms per milliliter PO to a culture dish.Then incubate the plates overnight at 37 degrees Celsius in a humidified incubator. Afterwards, aspirate the solution and wash the dishes three times with 1X PBS. Continue coating by adding 10 milliliters of PBS containing one microgram per milliliter mouse laminin 1 and two micrograms per milliliter fibronectin.Then, incubate the dishes as previously described. To begin, add 0.1%gelatin in PBS to a 10 centimeter dish. And leave it at room temperature for five minutes.As this plate is being coated, place frozen mouse embryonic fibroblasts, or MEFs in a 37 degree Celsius water bath to quickly thaw them. Next, aspirate the gelatin from the dish and plate MEFs at a density of approximately 50, 000 cells per square centimeter in DMEM supplemented with 10 percent fetal bovine serum. After incubating the dish overnight at 37 degrees Celsius aspirate the medium and wash the prepared MEF plate with 1X PBS.Proceed to add 10 milliliters of previously prepared hESC medium containing human pluripotent stem cells abbreviated as hPSCs, to a 10 centimeter dish. After seeding cells at a density of 5 million cells per dish incubate the plates at 37 degrees Celsius in five percent carbon dioxide. Proceed to feed the cells daily with 10 milliliters of fresh hESC medium.Prior to commencing differentiation inspect the plate for any colonies that appear to contain differentiated cells irregular borders, or transparent centers. Under a dissecting microscope in a laminar flow hood mechanically dislodge any such irregular colonies with a pipette. To perform coating with gelatinous protein such as Matrigel, first aliquot and freeze the protein in one milliliter parts in order to avoid repetitive freeze thaw cycles.Then, thaw and resuspend a one mililliter frozen aliquot with 19 milliliters of DMEM f12. Proceed to plate five milliliters of this mixture onto a 10 centimeter dish and incubate the dishes for one hour at RT.Afterwards, aspirate the gelatinous protein immediately before plating the cells and wash with DMEM F12. As this dish is being coated select a plate where hPSCs are at approximately 80%confluency.Proceed to aspirate the hESC medium. And then wash the cells with PBS. After removing the PBS, add three milliliters of 0.05%trypsin-EDTA to the plate.Proceed to vigorously shake the dish horizontially for two minutes until the MEFs are observed under a microscope to begin to lift off as single cells. During this time, expect the hPSC colonies to remain attached to the plate. Proceed to aspirate the trypsin once the MEFs have lifted but before the hPSC colonies have detached.Then, to the plate, add 10 milliliters of hESC medium containing 10 micromolar Y-27632 dihydrochloride a ROCK inhibitor. Afterwards detach the cells by pipetting up and down over the colonies. Next return to the dish with a gelatinous protein and aspirate the solution.Wash this dish with DMEM f12 to remove any clumps. And into it plate hPSCs at a 1 to 2 ratio. Then, to the plate, add hESC medium containing 10 micromolar Y-27632 dihydrochloride up to 10 milliliters.Incubate the cells at 37 degrees Celsius overnight. Feed the cells daily with ROCK inhibitor supplemented medium. When the hPSCs reach 80%confluency proceed to initiate neural differentiation and designate this time point as Day 0.To commence differentiation feed the cells on Day 0 and Day 1 with 10 milliliters of KSR differentiation containing 100 nanomolar LDN-193189 of the MP inhibitor and 10 micromolar SB 431542, an inhibitor of activin and Nodal. Continue on Day 2 by replacing the medium with that containing the same concentrations of the BNP and activin and Nodal inhibitors plus three micromolar CHIR99021, a Wnt Agonist. Then, on Day 3, feed the cells with KSR differentiation medium, supplemented with only 10 micromolar SB 431542 and three micromolar CHIR99021.After preparting a mixture of 75%KSR differentiaion medium and 25%N2 differentiation medium which also contains three micromolar of the Wnt Agonist. Feed the cells with 15 milliliters of this liquid on Day 4 and 5. Next, on Days 6 and 7 feed the cells with 15 milliliters of medium consisting of 50%KSR differentiation medium and 50%N2 differentiation medium which has also been supplemented with three micromolar CHIR99021 25 nanograms per milliliters BMP4 and 100 nanomolar EDN3.Then, on Days 8 and 9 feed the cells with 20 milliliters of 25%KSR differentiation medium and 75%N2 differentiation medium supplemented with the same concentrations of factors used on Days 6 and 7. Proceed to continue with neural differentiation on Day 10 by feeding the cells with 20 milliliters of N2 differentiation medium containing three micromolar CHIR99021 25 nanograms per milliliter BMP4 and 100 nanomolar EDN3. On Day 11, aspirate the solution from dishes previously coated with PO lamanin 1, and fibronectin.Then leave the plates uncovered in a tissue culture hood for approximately 10 to 15 minutes, allowing them to dry completely. Proceed to remove the medium from the Day 11 cells and wash them with PBS. Then, add four milliliters of cell detachment solution to the dish and incubate it at 37 degrees Celsius for 25 minutes.Following incubation add five milliliters of full melanocyte medium to the dish. And resuspend the cells by manually pipetting up and down until they have lifted off the plate. Transfer the suspension to a 15 milliliter tube and spin the cells down for five minutes at 200 Gs.After counting cells using Triypan Blue Exclusion and a hemocytometer, resuspend them in full melanocyte medium at a density of 2X10 to the 6th cells per milliliter.Plate 10 microliter droplets of the cell suspension onto the dried, coated dishes. Ensure that the droplets are close to one another but not touching, have well defined edges and do not run. To allow the cells to adhere, let the droplets stand at room temperature for 10 to 20 minutes.Afterwards, slowly add 10 milliliters of full melanocyte medium to the dish being careful not to disturb the attached cells. Then, move the dish to the incubator. Feed the cells every two to three days with full melanocyte medium.And expect to observe pigmentation in clusters of cells by the end of the first week. Once a week, passage cells by first associating them with cell detachment solution as previously described. After collecting and centrifuging the cells wash them twice with plain neurobasal medium.Then, replate the cells at a ratio of one to six in full melanocyte medium on dishes coated with PO, lamanin 1, and fibronectin. Around 30 days of differentiation the cells will appear deeply pigmented. Which is easily visualized when plated at high density or pelleted when passaging.Shown here is a bright field image of cells from a transgenic psox10 GFP human embryonic stem cell line cultured until Day 11 of the described differentiation protocol before cells are replated. Evident in these cultures are dark defined ridges of cells. When these cultures were assessed for GFP signal the dark ridges were enriched for sox10 positive cells.As sox10 is a transcription factor upregulated by neural crest stem cells and maintained in melanoblasts these results suggest that by Day 11 this differentiation method enriched for cells that will eventually give rise to melanocytes. As evidence of this, by Day 20 of the protocol cultures contained unpigmented, maturing, and fully mature pigmented melanocytes. At Day 25, the same types of cells are observed in cultures.The presence of maturing and pigmented melanocytes in these preparations are confirmed through staining with two melanin production proteins. TYRP2, which serves as a marker of both melanoblasts and melanocytes and TYRP1 which is a late stage melanocyte marker only expressed here in pigmented melanocytes. Once mastered, this protocol can be employed to differentiate human pluripotent stem cells into fully pigmented mature melanocytes in 25 days.The protocol proceeds through normal melanocyte development by first proceeding through the neuro crest state and as well as the melanoblast stage.

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Research

JoVE Journal

Developmental Biology

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Efficient Derivation of Retinal Pigment Epithelium Cells from Stem Cells

No cure has been discovered for age-related macular degeneration (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex multifactorial disease with an unknown etiology, although it is largely thought to occur due to death or dysfunction of the retinal pigment epithelium (RPE), a monolayer of cells that underlies the retina and provides critical support for photoreceptors. RPE cell replacement strategies may hold great promise for providing therapeutic relief for a large subset of AMD patients, and RPE cells that strongly resemble primary human cells (hRPE) have been generated in multiple independent labs, including our own. In addition, the uses for iPS-RPE are not limited to cell-based therapies, but also have been used to model RPE diseases. These types of studies may not only elucidate the molecular bases of the diseases, but also serve as invaluable tools for developing and testing novel drugs. We present here an optimized protocol for directed differentiation of RPE from stem cells. Adding nicotinamide and either Activin A or IDE-1, a small molecule that mimics its effects, at specific time points, greatly enhances the yield of RPE cells. Using this technique we can derive large numbers of low passage RPE in as early as three months.

Stem cell-derived retinal pigment epithelium (RPE) cells may be used for multiple applications including cell-based therapies for retinal degeneration, disease modeling, and drug studies. Here we present a simple protocol for reproducibly deriving RPE from stem cells.

No cure has been discovered for age-related macular degeneration  (AMD), the leading cause of vision loss in people over the age of 55. AMD is complex ...

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.