Name: efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction
Uploaded: 2011-11-03T13:00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction at JoVE.com

XHP has been supported by National Institute of Health (NIH) grants from National Institute on Aging (NIHK01AG024496) and The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NIHR21HD056530).

1. Solution and Media Preparation
<ol>
	<li>Gelatin coating solution: 0.1% (w/v) gelatin in ddH2O, autoclaved and store at 4&deg;C.</li>
	<li>Matrigel coating solutions. Stock solution: slow thaw Matrigel (10 ml) at 4&deg;C overnight, add 10 ml ice-cold DMEM or DMEM/F12, mix well and aliquot 1 ml/tube underneath sterilized tissue culture hood, store at -20&deg;C. Working solution: slow thaw 1 ml Matrigel aliquot at 4&deg;C for 1-2 hour, transfer to 14 ml chilled DMEM or DMEM/F12 and mix well underneath sterilized tissue culture hood immediately before coating.</li>
	<li>Human laminin coating solution: dilute 1 ml human laminin solution (0.5 mg/ml in Tris Buffered NaCl, store at -80&deg;C, slow thaw at 4&deg;C for 1-2 hour) with ice-cold DMEM or DMEM/F12 to 12.5 ml working solution (40 &mu;g/ml) underneath sterilized tissue culture hood immediately before coating.</li>
	<li>Growth factor stock solutions (500-1000X): dissolve growth factor at 10 &mu;g/ml in sterilized buffer (0.5% BSA, 1.0 mM DTT, 10% glycerol, 1XPBS) and store as 50-100 &mu;l/tube aliquots at -80&deg;C.</li>
	<li>HESC media: DMEM/F12 or KO-DMEM (80%), KO serum replacement (20%), L-alanyl-L-gln or L-gln (2 mM), MEM nonessential amino acids (MNAA, 1X), and &beta;-Mercaptoethanol (100 &mu;M), filtered and store at 4&deg;C, supplemented with 20 ng/ml bFGF before use. Or replacing "KO serum replacement" with defined components: DMEM/F12 or KO-DMEM (100%), L-alanyl-L-gln or L-gln (2 mM), MNAA (1X), MEM essential amino acids (MEAA, 1X), and &beta;-Mercaptoethanol (100 &mu;M), filtered and store at 4&deg;C, supplemented with bFGF (20 ng/ml), human insulin (20 &mu;g/ml), ascorbic acid (50 &mu;g/ml), human activin A (50 ng/ml), human albumin/Albumax (10 mg/ml), and human transferrin (8 &mu;g/ml) before use.</li>
	<li>HESC cardiac differentiation media: DMEM/F12 (90%), defined FBS (10%), and L-alanyl-L-gln or L-gln (2 mM).</li>
	<li>Media containing nicotinamide (NAM): add NAM to HESC media or HESC cardiac differentiation media to final concentration of 10 mM, filtered, store at 4&deg;C and use within 2 weeks of preparation.</li>
</ol>
2. Plate Coating
<ol>
	<li>Coat plates with gelatin: add 2.5 ml/well of gelatin solution to 6 well plates and incubate overnight in a 37&deg;C humidified incubator.</li>
	<li>Coat plates with laminin: chill gelatin-coated plates and remove gelatin, add 2.5 ml/well Matrigel or human laminin coating working solution to pre-chilled gelatinized 6-well plates and incubate at 4&deg;C overnight.</li>
</ol>
3. Passaging and Seeding Undifferentiated hESCs under Defined Conditions
<ol>
	<li>Allow hESC colonies grow to 5-7 days old, and take hESC culture plate to dissecting microscope (pre-warm dissecting stage to 37&deg;C) underneath dissecting sterilized hood.</li>
	<li>Select hESC colonies to be split. Morphologically, these colonies should have &gt; 75% undifferentiated hESCs (small compact cells), usually slightly opaque (not white-piled-up cells, not clear-differentiated cells) with defined edge underneath the dissecting microscope. Carefully outline the selected colonies and remove differentiated fibroblast layer surrounding the colony and all differentiated parts (if any) of the colony with the edge of P2 sterile pipette tip or pulled glass capillary.</li>
	<li>Remove the old media containing floating detached differentiated cells by aspirating. Wash with hESC media (without bFGF) once. Add 3 ml/well fresh hESC media containing 20 ng/ml bFGF.</li>
	<li>Cut the undifferentiated hESC colonies into small pieces, and detach with sterile pipette tip or pulled glass capillary.</li>
	<li>Pool the media containing detached colony pieces together in a 50 ml conical tube. Wash the plate once with 1 ml/well hESC media containing 20 ng/ml bFGF and pool together.</li>
	<li>Aspirate the Matrigel or human laminin solution from the coated fresh plates. Aliquot 4 ml/well hESC media containing colony pieces to a 6-well plate. Gently transfer the plate to incubator without shaking and allow colony pieces seed overnight without disturbing in a humidified 37&deg;C incubator with an atmosphere of 5% CO2.</li>
</ol>
4. Cardiac Induction of hESCs under Defined Culture System with Nicotinamide
<ol>
	<li>At day 3 after seeding, remove most of old media from each well of the plate and leave enough media to allow hESC colonies to be submerged (never allow hESCs to dry out). Replace with 4 ml/well fresh hESC media containing 20 ng/ml bFGF and 10 mM nicotinamide (NAM).</li>
	<li>Replace old media with fresh hESC media containing 20 ng/ml bFGF and 10 mM NAM every other day, and allow cardiac-induced hESC colonies grow to day 8-10. Upon exposing to NAM, all the cells within the colony will undergo morphology changes to large differentiated cells that will continue to multiply. The colonies will increase in size, and by day 8-10, and cells will begin to pile up in some areas of the colonies.</li>
</ol>
5. Continuing Cardiac Differentiation in Suspension Culture
<ol>
	<li>Take hESC culture plate to dissecting microscope (pre-warm dissecting stage to 37&deg;C) underneath dissecting sterilized hood. Carefully outline the colonies and remove fibroblast layer surrounding the colony (differentiated cells migrated out of the colony) with the edge of P2 sterile pipette tip or pulled glass capillary.</li>
	<li>Remove the old media containing floating detached fibroblast cells by aspirating. Wash with hESC media (without bFGF) once. Add 3 ml/well fresh hESC media (without bFGF).</li>
	<li>Cut the cardiac-induced hESC colonies into small pieces, and detach with sterile pipette tip or pulled glass capillary.</li>
	<li>Pool the media containing detached colony pieces together in a 50 ml conical tube. Wash the plate once with 1 ml/well hESC media (without bFGF) and pool together.</li>
	<li>Aliquot 4 ml/well serum-free hESC media containing colony pieces to a 6-well ultralow attachment plate and incubate in a 37&deg;C humidified incubator to allow floating cellular clusters (cardioblasts) to form for 4-5 days.</li>
</ol>
6. Beating Cardiomyocyte Phenotype Maturation in Adhesive Culture
<ol>
	<li>Pool the media containing floating cardioblasts together in a 50 ml conical tube. Centrifuge at 1400 rpm for 5 min. Aspirate the old media as much as you can and add equal amount of fresh hESC cardiac differentiation media containing 10 mM NAM.</li>
	<li>Pipette up and down to mix floating cardioblasts and aliquot 4 ml/well to 6-well plates. Transfer the plates to a 37&deg;C humidified incubator to allow cardioblasts to attach overnight.</li>
	<li>Replace hESC cardiac differentiation media containing 10 mM NAM every other day.</li>
	<li>At day 8, replace with hESC cardiac differentiation media without NAM, and continue to replace HESC cardiac differentiation media every other day. Beating cardiomyocytes will begin to appear in about 1-2 weeks of continuous cultivation after withdrawal of NAM and increase in numbers with time, and could retain strong and rhythmic contractions for over 3 months.</li>
</ol>
7. Representative Results:
Nicotinamide (NAM) is rendered sufficient to induce hESCs maintained in the defined culture system to transition from pluripotency exclusively to a cardiomesodermal phenotype (Fig. 2B). Upon exposure of undifferentiated hESCs to NAM, all the cells within the colony will undergo morphology changes to large differentiated cells that down-regulate the expression of Oct-4 and begin to express the cardiac specific transcription factor (Csx) Nkx2.5 and &alpha;-actinin, consistent with a cardiomesoderm phenotype (Fig. 2B). These differentiated cells will continue to multiply and the colonies increase in size. Increased intensity of Nkx2.5 will be usually observed in areas of the colonies where cells begin to pile up (Fig. 2B). After detached, the NAM-treated hESCs will form floating cellular clusters (cardioblasts) in a suspension culture to continue the cardiac differentiation process. After permitting the cardioblasts to attach and continuing to treat with NAM for 1 week, beating cardiomyocytes will begin to appear in about 1-2 weeks of continuous cultivation after withdrawal of NAM with a drastic increase in efficiency as compared to spontaneous multi-lineage differentiation of hESCs without treatment over the same time period (Fig. 2C). Cells within the beating cardiomyocyte clusters will express markers characteristic of cardiomyocytes, including Nkx2.5 and &alpha;-actinin (Fig. 2C). The contractions of the beating cardiomyocytes were confirmed by electrical profiles to be strong, rhythmic, well-coordinated, and well-entrained, with regular impulses reminiscent of the p-QRS-T-complexes seen from body surface electrodes in clinical electrocardiograms (Fig. 2C, also see the Video). The cardiomyocytes could retain their strong contractility for over 3 months.
<img alt="Figure 1" src="/files/ftp_upload/3274/3274fig1.jpg" /> 
Figure 1. Overall schemes of conventional approach versus small-molecule-induction approach for differentiation of human pluripotent stem cells towards specialized functional cells. (A) A schematic of conventional approach using multi-lineage inclination of human pluripotent stem cells through spontaneous germ layer differentiation. (B) A schematic of well-controlled efficient induction of human pluripotent stem cells exclusively to a particular clinically-relevant lineage by simple provision of small molecules.
<img alt="Figure 2" src="/files/ftp_upload/3274/3274fig2.jpg" /> 
Figure 2. Nicotinamide induces cardiac lineage specification direct of pluripotency under defined conditions and progression towards beating cardiomyocytes. (A) Schematic depicting of the protocol time line of directed cardiac differentiation of hESCs. (B) Upon exposure of undifferentiated hESCs to Nicotinamide (NAM) under the defined culture system, large differentiated Oct-4 (red) negative cells within the colony began to emerge, as compared to mock-treated (DMSO) hESCs as the control. NAM-induced Oct-4-negative cells began to express Nkx2.5 (green) and &alpha;-actinin (red), consistent with early cardiac differentiation. Progressively increased intensity of Nkx2.5 was usually observed in areas of the colony where cells began to pile up. All cells are indicated by DAPI staining of their nuclei (blue). (C) NAM-induced cardiac-committed hESCs yielded beating cardiomyocytes with a drastic increase in efficiency, as assessed by the cellular clusters that displayed rhythmic contractions (see electrophysiological profile and the Videos), and immunopositive for Nkx2.5 (green) and &alpha;-actinin (red). Phase images of representative large beating cardiomyocyte clusters and higher power images of representative contracting cardiac muscles are shown (also see corresponding videos). Arrows indicate the large beating cardiomyocyte cluster used for electrophysiological recording. Electrophysiological profile of the beating cardiomyocytes shows regular, entrained, rhythmic impulses reminiscent of the p-QRS-T-complexes seen in clinical electrocardiograms. Scale bars: 0.1 mm.
Videos: Contracting cardiomyocytes differentiated from NAM-induced hESCs. Videos 1 and 2 show representative large beating cardiomyocyte clusters in about 1 and 3 month after attachment. Video 3 and 4 show representative contracting cardiac muscles at higher power. Video 5 shows a typical small beating cardiomyocyte cluster spontaneously differentiated from hESCs without NAM treatment as control.
<a href="https://www.jove.com/files/ftp_upload/3274/3274video1.avi">Click here to watch Video 1.</a> 
<a href="https://www.jove.com/files/ftp_upload/3274/3274video2.avi">Click here to watch Video 2.</a> 
<a href="https://www.jove.com/files/ftp_upload/3274/3274video3.avi">Click here to watch Video 3.</a> 
<a href="https://www.jove.com/files/ftp_upload/3274/3274video4.avi">Click here to watch Video 4.</a> 
<a href="https://www.jove.com/files/ftp_upload/3274/3274video5.avi">Click here to watch Video 5.</a>

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The authors declare competing interests. XHP is the founder of Xcelthera. XHP and EYS have intellectual properties related to hESCs.

One of the major challenges for both developmental study and clinical translation has been how to channel the broad differentiation potential of pluripotent human stem cells to a desired phenotype efficiently and predictably. Although such cells can differentiate spontaneously in vitro into cells of all germ layers by going through a multi-lineage aggregate stage, in hESC-derived multi-lineage aggregates (embryoid bodies), only a very small fraction of cells (&lt;2%) spontaneously differentiate into cardiomyocytes13-15 (Fig. 1A). Following immunoselection, the enriched populations of cardiomyocytes were able to function as biological pacemakers to rescue the function of a damaged myocardium mechanically and electronically following injection into the heart of animal models13. In rodent infarcted models, engrafted hESC-derived cardiac progenies were able to survive and mature up to 12 weeks, and partially remuscularize the injured heart and improve the contractile function14,15. However, the inefficiency in generating human cardiac-committed cells through multi-lineage differentiation of pluripotent cells and the high risk of tumorigenicity following transplantation have hindered further clinical translation. Developing strategies to channel the wide differentiation potential of pluripotent hESCs exclusively and predictably to a cardiac phenotype is vital to harnessing the power of hESC biology in the heart field. In order to achieve uniformly conversion of human pluripotent stem cells to a particular lineage, we employed a defined culture system capable of insuring the proliferation of undifferentiated hESCs to identify conditions for well-controlled efficient induction of pluripotent hESCs exclusively to a particular clinically-relevant lineage by simple provision of small molecules (Fig 1B). We found that nicotinamide induced cardiac-lineage commitment direct from pluripotent hESCs under defined culture that further progressed to beating cardiomyocytes with high efficiency (Fig. 2). Nkx2.5 is an evolutionally conserved homeobox transcription factor indispensable for normal cardiac development and mutations of the gene are associated with human congenital heart disease (CHD), the most common human birth defect16. Expression of Nkx2.5 is the earliest marker for heart precursor cells in all vertebrates so far examined and is essential for proper cardiac septation and formation/maturation of electrical conduction system16. In vertebrates, the onset and pattern of early Nkx2.5 expression roughly coincide with the timing and area of cardiac specification in early embryogenesis, and Nkx2.5 gene continues to be expressed through development in the heart16. In our hESC model, NAM appeared to trigger the cardiac induction direct from pluripotent hESCs by promoting the expression of cardiac-specific transcription factor Nkx2.5 in a process that might emulate the specification of cardiomesoderm from the pluripotent epiblast in human embryonic cardiogenesis. Future studies will reveal genetic and epigenetic control molecules in human heart development as alternatives, which may pave the way for small molecule-mediated direct control and modulation of hESC pluripotent fate when deriving clinically-relevant lineages for regenerative therapies. Without NAM treatment, &lt; 2% hESCs will undergo spontaneous differentiation into beating cardiomyocytes12-15. With NAM treatment, we have been able to generate &gt; 95% embryonic cardiac precursors and &gt; 50% beating cardiomyocytes from hESCs maintained under a define culture in a process that might emulate human embryonic heart development12. Recently, known cardiac-fate determining genes have been used to transdifferentiate mouse fibroblasts into post-natal beating cardiomyocytes with a low efficiency of &lt; 0.5%17, 18. However, reprogrammed somatic cells have historically been associated with abnormal gene expression and accelerated senescence with impaired therapeutic utility19-21. Finally, the protocol we established here is limited to pluripotent hESCs derived from the inner cell mass (ICM) or epiblast of the human blastocyst4,5, may not apply to other pluripotent cells, including animal-originated ESCs, ESCs derived from earlier morula (eight-cell)-stage embryos22, and artificially reprogrammed cells23.

<table border="1">
<tbody>
<tr>
<th>Name of the reagent</th>
<th>Company	</th>
<th>Catalogue number</th>
<th>Comments (optional)</th>
</tr>
<tr>
<td>Gelatin</td>
<td>Sigma</td>
<td>G1890</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Matrigel</td>
<td>BD bioscience</td>
<td>356231</td>
<td>Growth factor reduced</td>
</tr>
<tr>
<td>Human laminin</td>
<td>Sigma</td>
<td>L6274</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Nicotinamide</td>
<td>Sigma</td>
<td>N0636</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM/F12</td>
<td>Invitrogen</td>
<td>10565042</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM</td>
<td>Invitrogen</td>
<td>31053036</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>DMEM-KO</td>
<td>Invitrogen</td>
<td>10829018</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Knock-out serum replacement</td>
<td>Invitrogen</td>
<td>10828028</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MEM nonessential amino acid solution 
(MNAA, 100X)		
</td>
<td>Invitrogen</td>
<td>11140050</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>MEM 
amino acids solution
(MEAA, 100X)		
</td>
<td>Invitrogen</td>
<td>11130050</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>Invitrogen</td>
<td>21985023</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Albumax</td>
<td>Invitrogen</td>
<td>11020021</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ascorbic acid</td>
<td>Sigma</td>
<td>A4403</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human transferrin</td>
<td>Sigma</td>
<td>T8158</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human bFGF</td>
<td>PeproTech</td>
<td>AF-100-18B</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human insulin</td>
<td>Invitrogen</td>
<td>12585014</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Human activin A</td>
<td>PeproTech</td>
<td>120-14E</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Defined FBS</td>
<td>Hyclone</td>
<td>SH30073-03</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>6-well ultralow attachment plate</td>
<td>Corning</td>
<td>3471</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>6-well plate</td>
<td>Corning</td>
<td>3516</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

Watch this Scientific Journal Video about Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction at JoVE.com

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

efficient-derivation-human-cardiac-precursors-cardiomyocytes-from

Research

JoVE Journal

Biology

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...