Name: chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity
Uploaded: 2017-02-24T12:30:00
Description: Watch this Scientific Journal Video about Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity at JoVE.com

This research was supported by a grant of the Korea Health Technology R&amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health &amp; Welfare, Republic of Korea (grant number: HI14C2116) and by Research Fund of Seoul St. Mary's Hospital, The Catholic University of Korea.

This study protocol was approved by the institutional review board of The Catholic University of Korea (KC16EAME0162) and performed according to NIH guidelines. All tissues were obtained with written informed consent.
1. Centrifugal Gravity Loading and Pellet Culture
<ol>
	<li>Cell culture and harvest
	<ol>
		<li>Culture ASCs (P2-P3; see List of Materials) in Dulbecco&#39;s Modified Eagle&#39;s Medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/stre...

<ol>
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N., de Crombrugghe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SOX9+is+a+potent+activator+of+the+chondrocyte-specific+enhancer+of+the+pro+alpha1(II)+collagen+gene.">SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene.</a> Mol Cell Biol. 17 (4), 2336-2346 (1997).</li><li>Jang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adipose+tissue-derived+stromal+vascular+fraction+for+clinical+application+to+cartilage+regeneration.">Characterization of adipose tissue-derived stromal vascular fraction for clinical application to cartilage regeneration.</a> In Vitro Cell Dev Biol Anim. 51 (2), 142-150 (2015).</li><li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+regeneration+of+articular+cartilage+and+subchondral+bone+in+vivo+using+MSCs+induced+by+a+spatially+controlled+gene+delivery+system+in+bilayered+integrated+scaffolds.">Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds.</a> Biomaterials. 32 (21), 4793-4805 (2011).</li><li>Janzen, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-cell+ageing+modified+by+the+cyclin-dependent+kinase+inhibitor+p16INK4a.">Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a.</a> Nature. 443 (7110), 421-426 (2006).</li><li>Muraglia, A., Cancedda, R., Quarto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+mesenchymal+progenitors+from+human+bone+marrow+differentiate+in+vitro+according+to+a+hierarchical+model.">Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model.</a> J Cell Sci. 113, 1161-1166 (2000).</li></ol>

The stemness state of cells is very important for CG-induced overexpression of SOX9. In our study, SOX9 expression could be induced by CG in early-passage ASCs (2-3), but not in later-passage ASCs. It has been reported that, during cultivation, ASCs contain CD34+ cells until 3 passages16. ASCs tend to lose the expression of CD34 as the cells are passaged, resulting in a low response to CG.
With centrifugal gravity force, hydrostatic pressure can be loaded onto cells dur...

Defects in articular cartilage do not heal naturally. Consequently, stem cell transplantation has been proposed as a promising approach for the repair of impaired cartilage. However, this method requires both the acquisition of a sufficient number of stem cells and the induction of these cells to undergo chondrogenic differentiation. Bone marrow (BM) has been widely used as a source of stem cells, but cell isolation from BM has two major disadvantages: invasiveness and insufficient yield. Because of its ease of acquisition, adipose tissue is a preferable source of stem cells. Previous studies demonstrated the feasibility of isolating stem cells from adipose tissue and inducing chondrogenic differentiation in these cells using cytokines, such as TGF-&#946;11,2. These methods are effective but expensive.
As a lower-cost alternative to cytokines, mechanical stress can be used to induce chondrogenic differentiation. Mechanical loading plays a critical role in maintaining the health of articular cartilage3, and it can induce chondrogenic phenotypes in various cells. For example, hydrostatic pressure induces chondrogenic phenotypes in synovium-derived progenitor cells via the MAP kinase/JNK pathway4, and mechanical compression induces chondrogenesis in human mesenchymal stem cells (MSCs) by upregulating chondrocytic genes5. In addition, shear stress contributes to the expression of chondrogenesis-related extracellular matrix (ECM) in human MSCs6. Centrifugal gravity (CG), an easily applied and controlled mechanical stress generated by centrifugation, can induce differential gene expression in cells7. For example, in lung epithelial carcinoma cells, the expression of interleukin (IL)-1b is upregulated by centrifugation8. Therefore, as an experimentally inducible mechanical stress, CG can be used to induce chondrocytic gene expression in stem cells. However, it remains unclear whether CG can induce the chondrogenic differentiation of stem cells.
In this study, we found that CG induced the upregulation of SOX9, a master regulator of chondrogenesis, in human ASCs, resulting in the overexpression of chondrocytic genes. In addition, we compared the effects of CG on chondrogenesis with those of TGF-&#946;1, the growth factor most commonly used to induce in vitro chondrogenesis in stem cells.

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			<td>50 mL Cornical Tube</td>
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			<td>10 mL Disposable Pipette</td>
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			<td>5 mL Disposable Pipette</td>
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			<td>ASC Culture Media Materials</td>
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			<td>DPBS</td>
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			<td>Chondrogenic Differentiation Media Materials</td>
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			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
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			<td>Dexamethasone</td>
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			<td>1%</td>
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			<td>Fetal Bovine Serum</td>
			<td>Life Technologies</td>
			<td>16000-044</td>
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			<td>Ascorbate-2-phosphate</td>
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			<td>A8960</td>
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			<td>Anti-Collagen II antibody</td>
			<td>abcam&#160;</td>
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			<td>1:100</td>
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			<td>&#160;Goat anti-Rabbit IgG (H+L) Secondary Antibody, 
			&#160;Alexa Fluor 594 conjugate&#160;</td>
			<td>Molecular Probe</td>
			<td>&#160;A-11037</td>
			<td>1:200</td>
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			<td>DAPI</td>
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			<td>Prolong gold antifade reagent</td>
			<td>Invitrogen</td>
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			<td>Slide Glass, Coated</td>
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			<td>Trizol</td>
			<td>Invitrogen</td>
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			<td>RevertAid First Strand cDNA Synthesis kit</td>
			<td>Thermo Scientfic</td>
			<td>K1622</td>
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			<td>i-Taq DNA Polymerase</td>
			<td>iNtRON BIOTECH</td>
			<td>25021</td>
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			<td>UltraPure 10X TBE Buffer</td>
			<td>Life Technologies</td>
			<td>15581-044</td>
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			<td>loading star</td>
			<td>Dyne Bio</td>
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			<td>Agarose</td>
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			<td>9012-36-6</td>
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			<td>1kb (+) DNA ladder marker</td>
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			<td>Human adipose-derived stem cells (ASCs)</td>
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chondrogenic differentiation induction of adipose-derived stem cells by centrifugal gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Centrifugal gravity induces the overexpression of chondrogenic differentiation markers in adipose-derived stem cells.
To determine the degree of centrifugal gravity force that is suitable to induce chondrogenic differentiation, ASCs were stimulated with different degrees of CG (0, 300, 600, 1,200, and 2,400 x g) for 15 min. After stimulation, the ASCs were re-seeded onto culture plates and cultured for 24 h. As shown in ...

Watch this Scientific Journal Video about Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity at JoVE.com

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

The overall goal of this procedure is to induce chondrogenic differentiation of ASC's for defective cartilage repair. This method is to induce the prechondrogenic differentiation of ASC's using centrifugal gravity force. Though this method can provide insight into chondrogenic differentiation of ASC's, it can also be applied to other systems, such as osteogenic and fibrogenic differentiation.The main advantage of this technique is that chondrogenic differentiation can be easily induced from stem cells without treating any cytokines. Demonstrating the procedure will be Yeri Alice Rim and Yoojun Nam from my laboratory. Begin by harvesting 80%confluent adipose-derived stem cells from a 100 milliliter culture dish by discarding the medium and washing with five milliliters of PBS.Then, add one milliliter of PBS containing one millimolar EDTA, and incubate for two minutes at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide. After two minutes, tap the culture plate gently then add four milliliters of fresh medium and transfer the cells to a 15 milliliter conical tube. Centrifuge the cells at 250 times g for two minutes at room temperature.After centrifugation, remove the supernatant without disturbing the pellet. Then, re-suspend the pellet in 10 milliliters of medium. Count the cells using a hemocytometer.For centrifugal gravity loading, transfer 2.5 times ten to the fifth cells to a new 15 milliliter conical tube, and centrifuge at 2400 times g for 15 minutes. Following the centrifugation, proceed to either pellet or micromass culture. Immediately after centrifugation, aspirate the supernatant and at 500 microliters of chondrogenic differentiation medium, or CDM, and 50 micrograms per milliliter of freshly-prepared L-ascorbic-acid 2-phosphate to the pellet.A positive control, induce chondrogenic differentiation and collect its cells by adding CDM containing 10 nanograms per milliliter TGF-beta-1. Loosely cap the tubes and incubate at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide for three weeks. Immediately after the gravity-loading centrifugation, aspirate the supernatant and re-suspend the pellet in 10 microliters of CDM.To form a micromass, pipette the re-suspended cells into the center of a well of a 24 well plate and then place the plate into the incubator. After two hours, carefully add 500 microliters of CDM to the well, pipetting against the wall of the plate to avoid disrupting the micromass. As a positive control, perform a micromass culture with uncentrifuged cells by adding CDM containing 10 nanograms per milliliter TGF-beta-1.Incubate the micromass for three weeks. On day 21 after micromass formation, use a pipette to harvest the spheroid pellets, and then wash with 1X PBS. Fix the spheroid pellets by immersion in 4%paraformaldehyde for 24 hours.The next day, discard the fixative and rinse the cell pellets with deionized water. Place one layer of gauze onto the tissue cassette and transfer the fixed spheroid pellets using a pipette. Cover the pellet by folding the gauze and close the cassette lid.Next, dehydrate the spheroid pellets by immersion in 70%ethanol for five minutes at room temperature. Then, continue through a series of ethanol concentrations for five minutes each. Then immerse the pellets in 100%xylene for 15 minutes.Immerse the spheroid pellets in paraffin at 56 degrees Celsius for embedding. Next, cut five micron-thick sections from the paraffin-embedded cell pellet using a rotary microtome. Float the sections in 50%ethanol, and then transfer them to a 50 degree Celsius water bath.Mount the floating sections onto slides, and then proceed to safranin o and alcian blue for immuno-flourescent staining. To determine a suitable centrifugal gravity force, ASC's were spun at centrifugal gravity of zero, 300, 600, 1, 200, and 2, 400 times g for fifteen minutes. SOX9 mRNA expression was evaluated at 24 hours after CG stimulation.A CG of 2, 400 times g significantly induced SOX9 mRNA expression. To evaluate the expression of chondrogenesis-related genes, total RNA was extracted from unstimulated ASC's, ASC's that had been centrifuged, or treated with TGF-beta-one. As seen here, CG stimulation induces collagen type-2 and aggrecan expression.over-expression of collagen type-2 in ASC's treated with CG was confirmed by immunoflourescence using an antibody against collagen type-2 in an Alexa Fluor 594 conjugated secondary antibody. To determine the effect of CG on chondrogenesis-related ECM over-expression, ASC's were treated with TGF-beta-one as a positive control. The negative control was a pellet culture not subjected to CG loading, or TGF-beta-one treatment.The over-expression of chondrogenesis-related ECM was evaluated in spheroid pellets consisting of centrifuged ASC's through the use of safranin o and alcian blue staining. Glycosaminoglycan, a major component of articular cartilage, was stained blue by alcian blue, or stained orange by safranin o. After 14 days of micromass culture, chondrocyte condensation was evaluated by the size of the aggregates.The size of each aggregate, generated by CG, was around 0.5 millimeters. Once mastered, this technique can be done in 30 minutes, if it is performed properly. After watching this video, you should have a good understanding of how induce chondogenic differentiation of stem cells using centrifugal gravity force.

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Research

JoVE Journal

Developmental Biology

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

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 ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...