Name: differentiation of human pluripotent stem cells into pancreatic beta-cell precursors in a 2d culture system
Uploaded: 2021-12-16T14:45:00
Description: Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System at JoVE.com

This work was funded by a grant from Qatar National Research Fund (QNRF) (Grant No. NPRP10-1221-160041).

The study has been approved by the appropriate institutional research ethics committee and performed following the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The protocol was approved by the Institutional Review Board (IRB) of HMC (no. 16260/16) and Qatar Biomedical Research Institute (QBRI) (no. 2016-003). This work is optimized for hESCs such as H1, H9, and HUES8. Blood samples were obtained from healthy individuals from Hamad Medical Cor...

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This work describes an enhanced protocol for generating pancreatic progenitors from hPSCs with a high co-expression of PDX1 and NKX6.1. Dissociation and replating of the hPSC-derived endoderm at half density on fresh matrix resulted in higher PDX1 and NKX6.1 in hPSC-derived pancreatic progenitors.
Although the growth factor cocktail for each stage is highly similar to P1-ND27, it has been shown that a more extended Stage 3 treatment including FGF and retinoid signaling ...

Diabetes is a complex metabolic disorder affecting millions of people globally. Supplementation of insulin is considered the only treatment option for diabetes. More advanced cases are treated with beta cell replacement therapy, achieved through transplantation of either whole cadaveric pancreas or islets1,2. Several issues surround transplantation therapy, such as limitation with the availability and quality of the tissue, invasiveness of transplantation procedures in addition to the continuous need for immunosuppressants. This necessitates the need for discovering novel and alternative options for beta cell replacement therapy2,3. Human pluripotent stem cells (hPSCs) have recently emerged as a promising tool for understanding human pancreas biology and as a non-exhaustive and potentially a more personalized source for transplantation therapy4,5,6,7. hPSCs, including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), have a high self-renewal capacity and give rise to any tissue type of the human body. hESCs are derived from the embryo&#39;s inner cell mass, and hiPSCs are reprogrammed from any somatic cell4,8.
Directed differentiation protocols are optimized to generate pancreatic beta cells from hPSCs that sequentially direct hPSCs through pancreatic developmental stages invitro. These protocols generate hPSC-derived islet organoids. While they have greatly improved at increasing the proportion of pancreatic beta cells therein, the efficiency of protocols is highly variable. It does not increase to more than ~40% of NKX6.1+/INSULIN+ or C-PEPTIDE + cells5,9,10,11,12,13. However, the generated beta cells are not entirely identical to the adult human beta cells in terms of their transcriptional and metabolic profiles and their response to glucose4,5,14. The hPSC-derived beta cells lack gene expression of key beta cell markers such as PCSK2, PAX6, UCN3, MAFA, G6PC2, and KCNK3 compared to adult humans islets5. Additionally, the hPSC-derived beta cells have diminished calcium signaling in response to glucose. They are contaminated with the co-generated polyhormonal cells that do not secrete appropriate amounts of insulin in response to increasing glucose levels5. On the other hand, hPSC-derived pancreatic progenitors, which are islet precursors, could be generated more efficiently in vitro compared to beta cells and, when transplanted in vivo, could mature into functional, insulin-secreting beta cells15,16. Clinical trials are currently focused on demonstrating their safety and efficacy upon transplantation in T1D subjects.
Notably, expression of the transcription factors PDX1 (Pancreatic and Duodenal Homeobox 1) and NKX6.1 (NKX6 Homeobox 1) within the same pancreatic progenitor cell is crucial for commitment towards a beta cell lineage5. Pancreatic progenitors that fail to express NKX6.1 give rise to polyhormonal endocrine cells or non-functional beta cells17,18. Therefore, a high co-expression of PDX1 and NKX6.1 in the pancreatic progenitor stage is essential for ultimately generating a large number of functional beta cells. Studies have demonstrated that an embryoid body or 3D culture enhances PDX1 and NKX6.1 in pancreatic progenitors where the differentiating cells are aggregated, varying between 40%-80% of the PDX1+/NKX6.1+ population12,19. However, compared to suspension cultures, 2D differentiation cultures are more cost-effective, feasible, and convenient for application on multiple cell lines5. We recently showed that monolayer differentiation cultures yield more than up to 90% of PDX1+/NKX6.1+ co-expressing hPSC-derived pancreatic progenitors20,21,22. The reported method conferred a high replicating capacity to the generated pancreatic progenitors and prevented alternate fate specifications such as hepatic lineage21. Therefore, herein, this protocol demonstrates a highly efficient method for the differentiation of hPSCs to pancreatic beta-cell precursors co-expressing PDX1 and NKX6.1. This method utilizes the technique of dissociating hPSC-derived endoderm and manipulating the cell density, followed by an extended FGF and Retinoid signaling as well as Hedgehog inhibition to promote PDX1 and NKX6.1 co-expression (Figure 1). This method can facilitate a scalable generation of hPSC-derived pancreatic beta-cell precursors for transplantation therapy and disease modeling.

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	<tbody>
		<tr>
			<td>15 mL, conical, centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>20X TBS Tween 20</td>
			<td>Thermo Scientific</td>
			<td>28360</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well culture plates, flat bottom with lid</td>
			<td>Costar</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL, conical, centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>6- well culture plates, multidish</td>
			<td>Thermo Scientific</td>
			<td>140685</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stem Cell Technologies</td>
			<td>0-7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Activin A</td>
			<td>R&#38;D</td>
			<td>338-AC</td>
			<td>Reconstituted in 4 mM HCl</td>
		</tr>
		<tr>
			<td>Anti NKX6.1 antibody, mouse monoclonal</td>
			<td>DSHB</td>
			<td>F55A12-C</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining</td>
		</tr>
		<tr>
			<td>Anti-PDX1 antibody, guinea pig polyclonal</td>
			<td>Abcam</td>
			<td>ab47308</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:1000 for immunostaining</td>
		</tr>
		<tr>
			<td>B27 minus Vit A</td>
			<td>ThermoFisher</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin, heat shock fraction, fatty acid free</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>Tocris</td>
			<td>4423</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>ThermoFisher</td>
			<td>41965047</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td></td>
			<td>A-21206</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X</td>
			<td>ThermoFisher</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>ThermoFisher</td>
			<td>PHG0313</td>
			<td>Reconstituted in 0.1% BSA in PBS</td>
		</tr>
		<tr>
			<td>FGF10</td>
			<td>R&#38;D</td>
			<td>345-FG</td>
			<td>Reconstituted in PBS</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8644</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33258</td>
			<td>Sigma</td>
			<td>23491-45-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut DMEM/F-12 (1X)</td>
			<td>Gibco</td>
			<td>12660-012</td>
			<td></td>
		</tr>
		<tr>
			<td>KnockOut SR serum replacement</td>
			<td>Gibco</td>
			<td>10828-028</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic acid (vitamin C)</td>
			<td>Sigma</td>
			<td>A92902</td>
			<td>Reconstituted in distilled water</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Aliquot the thawed stock and freeze at -20C.</td>
		</tr>
		<tr>
			<td>MCDB131</td>
			<td>ThermoFisher</td>
			<td>10372019</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SOX17</td>
			<td>ORIGENE</td>
			<td>CF500096</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:2000 for immunostaining</td>
		</tr>
		<tr>
			<td>mTeSR Plus</td>
			<td>Stem Cell Technologies</td>
			<td>85850</td>
			<td>Mix the basal media with supplement. Aliquot and store at -20 &#176;C for longer time or at 4 &#176;C for instant use</td>
		</tr>
		<tr>
			<td>Nalgene filter units, 0.2 &#181;m PES</td>
			<td>ThermoFisher</td>
			<td>566-0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>72340</td>
			<td>Reconstituted in distilled water</td>
		</tr>
		<tr>
			<td>NOGGIN</td>
			<td>R&#38;D</td>
			<td>6057-NG</td>
			<td>Reconstituted in 0.1% BSA in PBS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>ChemCruz</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Portable vacuum aspirator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-FOXA2</td>
			<td>Cell signaling technology</td>
			<td>3143</td>
			<td>Diluted to 1:100 for flow-cytometry and 1:500 for immunostaining</td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>Rock inhibitor (Y-27632)</td>
			<td>ReproCell</td>
			<td>04-0012-02</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>Round Bottom Polystyrene FACS Tubes with Caps, STERILE</td>
			<td>Stellar Scientific</td>
			<td>FSC-9010</td>
			<td></td>
		</tr>
		<tr>
			<td>SANT-1</td>
			<td>Sigma Aldrich</td>
			<td>S4572</td>
			<td>Reconstituted in DMSO</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>StemFlex</td>
			<td>ThermoFisher</td>
			<td>A3349401</td>
			<td>Mix the basal media with supplement. Aliquot and store at -20 &#176;C for longer time or at 4 &#176;C for instant use</td>
		</tr>
		<tr>
			<td>TALI Cellular Analysis Slide</td>
			<td>Invitrogen</td>
			<td>T10794</td>
			<td></td>
		</tr>
		<tr>
			<td>Tali image-based cytometer automated cell counter</td>
			<td>Invitrogen</td>
			<td>T10796</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>9002-93-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE 100 mL</td>
			<td>ThermoFisher</td>
			<td>12563011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA, pH 8.0</td>
			<td>Invitrogen</td>
			<td>15575-038</td>
			<td></td>
		</tr>
	</tbody>
</table>

differentiation of human pluripotent stem cells into pancreatic beta-cell precursors in a 2d culture system

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The results show that optimized protocol P2-D (Figures 1A) enhanced pancreatic progenitor differentiation efficiency by upregulating PDX1 and NKX6.1 co-expression (Figure 2A,B, and Figure 3A). In particular, the results showed that dissociation of endodermal cells and their replating on fresh membrane matrix along with a longer duration of Stage 3 enhanced NKX6.1 expression in hPSC-derived pancreatic progenitors (op...

Watch this Scientific Journal Video about Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System at JoVE.com

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

This optimized protocol enhanced the generation of pancreatic beta cell precursors from human pluripotent stem cells, which can be used for cell therapy for diabetes and investigating molecular mechanisms underlining diabetes biogenesis. This is a feasible technique as it is used for 2D monolayer cultures. It is easy to apply, and is consistent across multiple control and patient-derived pluripotent stem cell lines.These beta cell precursors when transplanted in a diabetic patient can mature into insulin secreting beta cells, and can potentially reverse hyperglycemia. Therefore, the increased percentages of pancreatic progenitors generated using our protocol can be used for cell therapy for diabetes. This enhanced protocol can be used for studying early human pancreatic development in healthy individuals as well as in diabetic patients, which is challenging due to a shortage of tissue samples.When the human pluripotent stem cell or hPSC colonies have reached 70 to 80%confluence, wash them twice with warm PBS inside the tissue culture hood. Then aspirate the buffer using a portable vacuum aspirator. Next, add stage one differentiation medium containing CHIR-99021 to the colonies, and incubate the plate at 37 degrees Celsius for 24 hours.The next day, replace the spent media with stage one differentiation medium without CHIR-99021. After aspirating the spent medium and washing the definitive endoderm cells twice with warm PBS, swirl the plate to remove any cell debris. Then fix the cells by adding 4%paraformaldehyde, and place the plate on a two-dimensional shaker.After fixation, wash the cells with TRIS-buffered saline containing 0.5%Tween, and place the plate on the shaker. Then permeabilize the fixed cells by adding a generous amount of PBS containing 0.5%Triton X-100 and place the plate back on the shaker. Next, add freshly prepared blocking buffer to the permeabilized cells, and incubate the plate for one hour on the shaker.Then add diluted primary antibodies to the blocked cells, and place the plate on the shaker at four degrees Celsius with gentle shaking. The following day after aspirating the primary antibody solution, wash the wells three times with TBST for 10 minutes each on the shaker. Next, add the secondary antibodies.Cover the plate with aluminum foil to protect from light, and place the plate on the shaker at room temperature for one hour. Then aspirate the secondary antibody solution, and wash the stained wells with TBST on the shaker for 10 minutes, keeping the plate covered with foil. Next, to stain the nuclei, add Hoechst solution to the wells and place the plate on the shaker.After two to three minutes, aspirate the Hoechst solution and rinse the wells twice with PBS. Finally, add PBS to the stained cells, and image them using an inverted fluorescence microscope in the dark. On day one of stage two, dissociate the hPSC derived endodermal cells by first washing them with warm PBS, and then adding one milliliter of warm enzyme solution per well of a six well plate.Place the plate at 37 degrees Celsius and 5%carbon dioxide for three to five minutes or until the cells begin to detach from one another. Dissociate the detached sheets or monolayer of cells in the wells, and collect the detached cells together in a 15 milliliter polypropylene tube using basal stage half media. Then spin the cells down, discard the supernatant, and resuspend the pellet using one milliliter of sterile PBS.After counting the cells using an automated counter, spin the cells again, and discard the supernatant. Then resuspend the pellet in the appropriate volume of stage two differentiation medium containing a Rock inhibitor. Plate the resuspended cells on one to 50 membrane matrix coated plates and incubate them at 37 degrees Celsius and 5%carbon dioxide.After 24 hours, replace the media with stage two differentiation medium without the Rock inhibitor. At the end of stage four, detach cells as demonstrated earlier, and collect them in a 15 milliliter tube. Then spin the cells, discard the supernatant, wash the cells with PBS, and dissociate them into single cells.After counting the cells using an automated cell counter, spin the cells again and resuspend the pellet in 200 microliters of chilled PBS. Next, add two milliliters of chilled 80%ethanol drop wise with the tube on a vortex set low to medium speed. Close the cap tightly, and place the tube slightly tilted on the shaker at four degrees Celsius overnight.The next day, spin the cells and wash the pellet with PBS to dissociate any clumps of fixed cells. Then block the fixed cells for at least one hour at room temperature or four degrees Celsius overnight on the shaker. Next, to stain for the pancreatic progenitor markers, distribute 200, 000 cells per condition including appropriate isotype controls, unstained, and secondary antibody controls in a 96 well V bottom plate.Then spin the plate briefly, and flip the plate with a swift motion to discard the supernatant without losing the pellets. Next, incubate the cells in primary antibodies for at least two hours at room temperature on a shaker with gentle shaking. Then wash the stained cells three times with TBST by pipetting up and down in the wells.Centrifuge the cells again, discard the supernatant, and add secondary antibodies to the cells. After a 30 minute incubation at room temperature, wash the stained cells twice with TBST. Then spin the plate, collect the stained cells in 100 microliters of PBS in light protected fax tubes, and run the samples on a flow cytometry machine.Compared to the non-optimized method, the optimized protocol enhanced pancreatic progenitor differentiation efficiency by upregulating PDX 1 and NKX 6.1 co-expression. In particular, the disassociation of endodermal cells and their replating on fresh membrane matrix, along with a longer duration of stage three, enhanced NKX 6.1 expression in hPSC derived pancreatic progenitors compared to the non-dissociated protocol. Pancreatic progenitors generated using the optimized protocol also increased numbers of SOX9 positive cells compared to the non-dissociated method.Further, the optimized method also generated a higher proportion of proliferative NKX 6.1 positive cells that co-expressed the proliferation marker Ki67. In order to enhance the efficiency, it is crucial to dissociate the HbA1 derived endoderm and then extend the duration of stage three treatment with FGF amyloid signaling and Hedgehog inhibition. Scalable generation of pancreatic progenitors using this enhanced protocol has facilitated studies on improving efficiency and maturation of human pluripotent stem cell derived pancreatic beta cells and allowed modeling of different type of diabetes.

differentiation-human-pluripotent-stem-cells-into-pancreatic-beta

Research

JoVE Journal

Developmental Biology

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

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 ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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

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

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...