Name: integration free derivation of human induced pluripotent stem cells using laminin 521 matrix
Uploaded: 2017-07-07T15:00:00
Description: Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

This work was supported by SSF (B13-0074) and VINNOVA (2016-04134) funding agents.

The collection of patient material and derivation of hiPS cells is approved by the Ethics Review Board, Stockholm, March 28, 2012, Registration number: 2012/208-31/3. Cell culture steps should be performed in biosafety cabinets unless otherwise mentioned. Always practice sterile handling techniques when working with cells. Allow media, plates and reagents to reach room temperature before starting. Incubate cells at 37 &#7506;C, 5% CO2 in high humidity.
1. Isolation of Human Fibroblast...

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The expected result of this protocol is the successful generation of several clonally derived hiPS cell lines. Importantly, the method for the maintenance of and expansion of established hiPS cells here described is reliable and can be performed with little prior experience of stem cell culture. Enzymatic single cell passaging with ROCKi together with the LN-521 matrix is known to maintain cells as karyotypically normal, pluripotent and readily able to differentiate while avoiding induced heterogeneity that colony based ...

Since the first derivation of hiPS cell lines by Takahashi et al.1,2, hiPS cells have provided a useful tool for disease modeling, drug discovery and as source material for generating cell therapies in regenerative medicine3. hiPS cell culture has long been dependent on co-culture with fibroblast feeder cells4,5 or on Matrigel6 and with media formulations containing fetal bovine serum (FBS). Batch-to-batch variances are a common consequence of the undefined nature of these culture conditions, resulting in unpredictable variations, which is a major contributor to the unreliability of these protocols7. The development of defined medium such as Essential 8 (E8)8 and defined cell culture matrices for instance LN-5219, allows for the establishment of highly reproducible protocols and aid in the robust generation and maintenance of homogenous hiPS cells7,8,9,10.
Development of integration free reprogramming techniques have been a leap forward. Originally, reprogramming depended on retroviral vectors which randomly integrated into the genome with disruptive effects on genomic integrity11. Advances in reprogramming methodologies includes the development of RNA based vectors. RNA vectors have an advantage over the DNA based reprogramming method as unintended integration through genomic recombination is not possible12. SeV vectors provide high and transient expression of exogenous factors through single-stranded RNA without a DNA-phase11. The reprogramming vectors delivered by the SeV are diluted throughout cell expansion and eventually shed from culture providing a foot-print free way of reprogramming. Thereafter, maintenance of pluripotency is dependent on endogenous expression of the pluripotency genes2.
As pioneering hiPS cell based therapies are beginning to move into clinical trials, the demands for standardized batches, reproducibility, and safety are essential issues to address13. Therefore, products of animal origin should be avoided. For instance, the use of xenogeneic products has been associated with risk of nonhuman pathogen contamination. Also, cells cultured in the presence of animal derived culture components have been shown to incorporate nonhuman siliac acids into cell membranes which threatens to render derived cells immunogenic14. Hence, the need to eliminate xenogeneic products is necessary to any future clinical pursuits. This protocol applies xeno-free and defined culture in the maintenance of hiPS cells moving cells closer to clinical compliance.
This protocol describes a consistent, highly reproducible and easy-to-use method that generates standardized hiPS cells from fibroblasts. It also offers a user-friendly culture system for the maintenance of established hiPS cells. This protocol has been used to derive more than 300 hiPS cell lines in the Swedish national human iPS Core facility at Karolinska Institutet of which some lines have previously been described15,16.

<table border="0" cellpadding="0" cellspacing="0" width="711">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Dispase</td>
			<td style="width:155px;">Life Technologies</td>
			<td style="width:119px;">171105-041</td>
			<td style="width:223px;">Biopsy digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma</td>
			<td>C0130</td>
			<td>Biopsy digestion</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td>Fibroblast matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IMDM</td>
			<td>Life Technologies</td>
			<td>21980002-032</td>
			<td>Fibroblast medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Invitrogen</td>
			<td>10270106</td>
			<td>Fibroblast medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PenicillinStreptomycin</td>
			<td>Life Technologies</td>
			<td>15070-063</td>
			<td>Antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Essential 8 media</td>
			<td>ThermFisher Scientific</td>
			<td>A1517001</td>
			<td>iPS cell culture media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LN-521</td>
			<td>Biolamina</td>
			<td>LN521-03</td>
			<td>iPS cell culture matrix</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TrypLE select 1X</td>
			<td>ThermoFisher Scientific</td>
			<td>12563011</td>
			<td>Dissociation reagent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rho-kinase inhibitor Y27632</td>
			<td>Millipore</td>
			<td>SCM075</td>
			<td>Rho-kinase inhibitor (ROCKi)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CytoTune &#8211; iPS 2.0 reprogramming kit</td>
			<td>Life Technologies</td>
			<td>A1377801</td>
			<td>Sendai virus reprogramming vector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm tissue culture dish</td>
			<td>Sarstedt</td>
			<td>83.3900.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60 mm tissue culture dishes</td>
			<td>Sarstedt</td>
			<td>83.3901.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 well tissue culture plates</td>
			<td>Sarstedt</td>
			<td>83.3922.300</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T25 tissue culture flasks</td>
			<td>VWR</td>
			<td>734-2311</td>
			<td>Cell culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td>430791</td>
			<td>Centrifuge tubes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030123.301</td>
			<td>1.5 ml tube</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CoolCell cell LX</td>
			<td>Biocision</td>
			<td>BCS-405</td>
			<td>Freezing container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMSO</td>
			<td>Sigma</td>
			<td>D2650-100ml</td>
			<td>Fibroblast freezing</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Cryovials 1.8 ml</td>
			<td>VWR</td>
			<td>479-6847</td>
			<td>Cryovials</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PSC Cryopreservation Kit</td>
			<td>Gibco</td>
			<td>A2644601</td>
			<td>PSC CryomediumRevitaCell supplement</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin-EDTA (0.05%)</td>
			<td>ThermoFisherScientific</td>
			<td>25300054</td>
			<td>Fibroblast dissociation enzyme</td>
		</tr>
		<tr height="20">
			<td height="40" style="height:40px;">DMEM/F12</td>
			<td>Life Technologies</td>
			<td>31331-028</td>
			<td>Digestive enzymes dilutant</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS</td>
			<td>ThermoFIsher Scientific</td>
			<td>14025-050</td>
			<td>Biopsy preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Haemocytometer</td>
			<td style="width:155px;">Sigma</td>
			<td style="width:119px;">BRAND, 718920</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

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.

From biopsy to hiPS cells
The entire process from biopsy to established hiPS cells, clear of reprogramming vector and ready for characterization, takes approximately 16 weeks (Figure 1). A more detailed timeline is specified in Figure 2A. Approximately 4 weeks is needed to establish and expand fibroblast cultures. The first hiPS cell colonies started to emerge about three-...

Watch this Scientific Journal Video about Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix at JoVE.com

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

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) ...

The overall goal of this protocol is to derive homogenous human induced pluripotent stem cells. This method can be used to derive human induced pluripotent stem cells for many purposes including disease modeling, drug screening, or to derive cells for preclinical cell therapy trials. The advantage of this protocol is that it's robust and generates homogenous cells that are maintained in a user-friendly, Xeno-free, and fully chemically defined culture system.Since human iPS cells recapitulate the genetic background of the donor tissue and can potentially be differentiated into any disease-relevant cell type, they provide excellent tools for thesis modeling. The biopsy dissection and colony picking are intricate procedures. Seeing them performed with experienced hands assists in the understanding of the procedures and makes it easier to repeat.Demonstrating the procedure today will be Harriet RonnHolm and Kelly Day, two highly experienced laboratory technicians from the iPS Core Facility at Karolinska Institutet. Skin biopsy samples can be stored in PBS plus 1%penicillin streptomycin for up to 48 hours at four degrees Celsius, but should be processed as fast as possible after being obtained. Transfer the biopsy to a 35 millimeter dish.Submerge the skin biopsy in a 35 millimeter dish with two milliliters of 70%ethanol for 30 seconds. Add one milliliter of sterile Hank's Balanced Salt Solution supplemented with 1%penicillin streptomycin to a new 35 millimeter dish and transfer the biopsy to the new dish. Transfer the biopsy to a four 35 millimeter dish filled with one milliliter of freshly-prepared 0.1%dispase solution.Using sterile surgical scalpels and forceps, cut the skin biopsy into one to two cubic millimeter pieces. Transfer the biopsy pieces with the dispase solution into a 15 milliliter tube and add an additional two milliliters of dispase. Rinse the 35 millimeter dish with one milliliter of dispase solution and transfer the rinsed solution to the 15 milliliter tube.Incubate the biopsy at four degrees Celsius overnight. On the following morning, add four milliliters of 0.1%collagenase I to the tube with the biopsy pieces and incubate at 37 degrees Celsius for four hours. Thirty minutes prior to seeding the cells, coat one well in a six-well tissue culture plate with one milliliter of 0.1%gelatin in DPBS.Incubate at room temperature for 30 minutes. After four hours of incubation with collagenase, centrifuge the digested cells at 300 times g for three minutes at room temperature. Aspirate the supernatant and resuspend the digest in two milliliters of fibroblast medium.Remove the gelatin solution from the previously prepared six-well tissue culture plate. Transfer the cell suspension including any remaining tissue pieces to the six-well plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide in high humidity.Fibroblasts are ready to be passaged to a T25 tissue culture flask when 80%confluent. Fibroblasts at passage two to four are optimal for reprogramming since reprogramming efficiency is generally higher for lower passage fibroblasts. Aspirate the cell culture medium from the flask and wash the cells with one milliliter of DPBS.Aspirate the DPBS and add one milliliter of 0.5%Trypsin EDTA. Incubate at 37 degrees Celsius for five minutes or until cells are detached. Add two milliliters of fibroblast medium, resuspend the cells, and transfer the cell suspension to a 15 milliliter tube.Centrifuge at 300 times g for three minutes at room temperature. Aspirate the supernatant and resuspend the pellet in two milliliters of fibroblast medium. After counting the fibroblasts using a hemacytometer, seed fives times 10 to the fourth cells in one well of a 24-well tissue culture plate.Incubate cells at 37 degrees Celsius overnight. On the following day, because Sendai virus will be handled, continue the procedure in a designated BSL-2 laboratory while using the appropriate personal protective equipment. Aspirate the cell culture medium and add 250 microliters of Sendai virus solution.Incubate the plate at 37 degrees Celsius. One day before picking the colonies, prepare LN-521 coated 24-well tissue culture plates. Pipette 250 microliters of LN-521 solution into one well of a 24-well plate.Seal the plate using parafilm and incubate at four degrees Celsius overnight. On the following day, aspirate the LN-521 solution from the one well of the 24-well plate and add 500 microliters of E8 into the well. The use of a hairnet and procedure mask is recommended while picking colonies under the stereo microscope.Colonies are ready to be picked when they reach a size of greater than one millimeter in diameter, display sharp edges, and are growing in homogenous monolayers. Do not pick colonies with fuzzy borders and large uncompact heterogeneous cell masses. Cut each colony with a scalpel into smaller pieces in a grid-like pattern.Use a 200 microliter pipette to mechanically scrape the cell sheets from the dish and then transfer the sheet to a well of a 24-well tissue culture plate previously coated with LN-521. Colony selection is critical for the success of this protocol. Picking a few additional colonies will ensure attachment in sufficient numbers.Allow the cells to attach without changing the E8 medium for 48 hours. Enzymatically passage the cells when the colonies have reached a size of greater than five millimeters. Aspirate the cell culture medium from the cells and wash with 250 microliters of DPBS.Aspirate the DPBS and add 250 microliters of dissociation reagent. Incubate at 37 degrees Celsius for three minutes. Observe that the cells have started to round up.Detach cells from the plate by rinsing the cells with dissociation reagent. Add 500 microliters of E8 medium to a 15 milliliter tube and transfer the cells and the dissociation reagent to the tube. Centrifuge at 300 times g for three minutes.Aspirate the supernatant from the 15 milliliter tube and resuspend the cell pellet in 500 microliters of E8 medium. Count the cells using a hemacytometer and seed 2.5 to five times 10 to the fourth cells in another previously prepared LN-521 precoated well containing 500 microliters of room temperature E8 medium. Add ROCK inhibitor to a final concentration of 10 micromolar.Incubate the cells at 37 degrees Celsius. Using this protocol, human induced pluripotent stem cell colonies emerge from transduced fibroblasts at about day 12 post-transduction. Colonies were ready to be picked at about three to four weeks post-transduction.Preferred features include flattened compact cell masses, growth as homogenous monolayers, and sharp edges. These bright field images show an early passage homogenous fully reprogrammed human iPS cell line compared to a highly heterogeneous cell line. The loss of the Sendai virus vector was confirmed by the absence of Sendai virus vector-specific markers in human iPS cells at passage 12 in contrast to human iPS cells at passage three.Immunocytochemistry staining of derived cells for pluripotency markers OCT4, SSEA-4, and NANOG yielded a homogeneously positive result. mRNA expression of endogenous pluripotency genes was shown by reverse transcriptase PCR. Cell cultures containing partially reprogrammed cells that did not express pluripotency factors were discarded.The differentiation potential of human iPS cells was assessed by formation of embryoid bodies. Real-time PCR after 21 days of embryoid body differentiation detected mRNA expression of endoderm, mesoderm, and ectoderm-specific markers which confirmed the derived human iPS cells were capable of differentiation to the three germ layers. Once mastered, this technique can be done with about 22 hours of hands-on time during a time period of approximately 16 weeks.The derived human induced pluripotent stem cells can be differentiated into any cell type of the adult human body to answer any questions relevant to your research field. After watching this video, we hope that you will have a good understanding of how to perform the steps necessary to derive induced pluripotent stem cells from skin biopsies.

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Research

JoVE Journal

Developmental Biology

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 ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

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 ...

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 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 ...

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

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 ...

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 ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...