generating single positive cd8 t cells from induced pluripotent stem cells

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

Prepare gelatinized OP9-delta-1 dishes one week prior to co-culture with human IPSCs. Add 4 milliliters of 0.1% gelatin to three new cell culture dishes, and incubate them for 30 minutes at 37 degrees Celsius. After the incubation, aspirate the gelatin and add 8 milliliters of OP9 media to each dish. Passage one confluent dish of OP9-delta-1 cells to three gelatin-coated dishes.
After four days, add 10 milliliters of OP9 media to each dish of cells for a total of 20 milliliters of media per dish. Begin human IPSC co-culture on OP9-delta-1 confluence dishes seven to eight days after passaging the cells. Aspirate the media from a confluent dish of human IPSCs, and add 10 milliliters of OP9 media.
Cut and detach cell colonies using a disposable cell passaging tool, and transfer 350 to 600 clumps of cut colonies onto a pregelatinized OP9-delta-1 dish with 10 milliliters of fresh OP9 media. Rock the culture dish side-to-side and front-to-back to ensure even distribution of colonies. The next day, aspirate the spent media, and replace it with 20 milliliters of fresh OP9 media.
The human IPSC clumps co-cultured on OP9-delta-1 for one day should appear as small, round monolayer colonies. On day 5, aspirate 10 milliliters of spent media and add 10 milliliters of fresh OP9 media. The colonies will begin to differentiate into primitive mesoderm, which is characterized by a multilayered dark center.
Repeat this process on day 9, at which point the multilayer center structures will evolve into dome-like shapes. Harvest the hematopoietic progenitor cells on day 13, at which point the structures are composed of a dark central organoid surrounded by a network of dome-like areas. Aspirate the media, and wash the cells once with 5 milliliters of 1x phenol red-free Hank&#39;s balanced salt solution with calcium and magnesium or HBSS.
After the wash, add 250 microliters of 5,000 units per milliliter collagenase-4 to 10 milliliters of HBSS. Add the mixture to the cells and incubate them at 37 degrees Celsius for 45 minutes. Aspirate the HBSS-collagenase mixture, and wash the cells once with 5 milliliters of PBS. After washing with PBS, add 5 milliliters of 0.25% trypsin-EDTA, and incubate the cells at 37 degrees Celsius for 20 minutes.
Then, add 4 milliliters of OP9 media, and associate the cell layer by pipetting to make a single-cell suspension. Transfer the cell suspension to a 50-milliliter conical tube through a 100-micrometer strainer, and centrifuge the tube at 300 times g and 4 degrees Celsius for 5 minutes. Aspirate the supernatant, and resuspend the cells in 10 milliliters of OP9 media.
Plate the cell suspension on a new gelatinized 10-centimeter cell culture dish, and incubate them at 37 degrees Celsius for 45 minutes. Then, collect any nonadherent cells by gentle pipetting. Transfer the cell suspension to a 50-milliliter conical tube through a strainer, and centrifuge as previously described. Then, aspirate the supernatant, and resuspend the cells in 10 milliliters of differentiation media.
Plate the cell suspension onto a new OP9-delta-1 confluent dish. Passage the cells on day 16 according to the manuscript directions, and continue passaging the nonadherent cells every five to seven days thereafter. After 35 days of differentiation, enrich the CD4-positive cell population by CD4 magnetic bead isolation according to the manufacturer&#39;s protocol.
Then, resuspend the cells in OP9 media and plate 1 milliliter of cell suspension into each well of a tissue culture flat-bottom 24-well plate of confluent OP9-delta-1. Stimulate cells by adding 100 international units of human IL-2, 5 nanograms of human IL-7, 500 nanograms of anti-human CD3 antibody, and 2 micrograms of anti-human CD28 antibody to each well. After four to seven days, cells can be collected for further analysis.

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Nanyang Technological University

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1. Culturing Human iPSCs (hiPSCs) on Mouse Embryonic Fibroblasts (MEF)
NOTE: Alternative methods for culturing hiPSCs can also be used, including but not limited to seeding onto a 6-well plate pre-coated with gelatin, a gelatinous protein mixture, recombinant laminin 511, or any other extracellular matrix used in hiPSC expansion, and cultured using defined media specially formulated for human pluripotent stem cell culture.
<ol>
	<li>Culturing MEF
	<ol>
		<li>Coat a 10 cm cell culture Petri dish with 4 mL of 0.1% gelatin and incubate for 30 min at 37 &#176;C.</li>
		<li>Thaw a vial of 4 x 106 irradiated MEF quickly into 10 mL of 37 &#176;C MEF media (Dulbecco&#39;s Modified Eagle Medium [DMEM] + 10% fetal bovine serum (FBS) + 1x penicillin-streptomycin + 1x L-glutamine supplement). Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend the cell pellet in 9 mL of MEF media.</li>
		<li>Remove the gelatin-coated dish from the incubator. Aspirate gelatin and add 7 mL of MEF media. Plate 3 mL of MEF suspension (from step 1.1.2) onto the gelatin-coated dish. Rock the dish side-to-side and front-to-back to ensure even distribution of MEF over the dish. Incubate at 37 &#176;C for 8-36 h.</li>
	</ol>
	</li>
	<li>Passaging hiPSC on MEF 
	NOTE: The data was generated using melanoma antigen recognized by T cells 1 (MART-1) iPSC derived from long-term cultured melanoma tumor-infiltrating lymphocyte (TIL), which specifically recognizes MART-1 peptide in the context of human leukocyte antigen (HLA)-A*02:01.
	<ol>
		<li>Passage hiPSCs when colonies are between 0.8 - 1.2 mm in diameter. Before passaging, check hiPSC colonies in a stereo-microscope and remove any areas of differentiation from the culture using the plastic edge of a 200 &#181;L tip.</li>
		<li>Aspirate spent media and add 10 mL hiPSC media (human embryonic stem cell [ES] culture media [Table of Materials] + 10 ng/mL human basic fibroblast growth factor [hbFGF]) supplemented with 10 &#956;M Rho-associated protein kinase (ROCK) inhibitor.</li>
		<li>Hold the cell culture dish in one hand and roll a disposable cell passaging tool across the entire dish in one direction. Apply enough pressure so that the entire roller blade touches the culture dish and maintains uniform pressure during the rolling action.</li>
		<li>Rotate the culture dish at 90&#176; and repeat step 1.2.3. View the plate in the microscope to visually confirm the proper cutting of the colonies, which should appear checkered. Detach cut colonies by gentle mechanical flushing using a 200 &#956;L pipette. 
		NOTE: Detachment of cut colonies by mechanical flushing must be done immediately after cutting colonies with the roller because after 3 min, the cut colonies will start to reattach to the dish, and it will become difficult to detach colonies of homogeneous size by flushing.</li>
		<li>Transfer 350 - 600 clumps of cut colonies onto a new 10 cm dish of MEF (plated 8 - 36 h before hiPSC passaging) with 10 mL of fresh hiPSC media supplemented with 10 &#956;M ROCK inhibitor. Incubate at 37 &#176;C. 
		NOTE: 600 clumps represent approximately 1.0 x 106 MART-1 iPSC and will yield 0.5-1.0 x 106 DP cells on day 35. However, expected numbers will vary depending on the potency of the starting cell line and culture conditions.</li>
		<li>The following day, aspirate spent media and add 10 mL of fresh hiPSC media. Change hiPSC media every 1-2 days, depending on the hiPSC growth rate.</li>
	</ol>
	</li>
</ol>
2. Preparation of OP9/DLL1 Cells for Co-culture with hiPSCs
<ol>
	<li>Culture OP9/DLL1 cells in OP9 media [&#945;-minimum essential medium (&#945;-MEM) + 20% fetal bovine serum (FBS) + 1x penicillin-streptomycin] at 37 &#176;C. When OP9/DLL1 cells reach confluency, aspirate media and wash once with 5 mL of 1x magnesium, calcium, and phenol red-free phosphate-buffered saline (PBS).</li>
	<li>Aspirate PBS and add 2 mL of 0.05% Trypsin-ethylenediaminetetraacetic acid (EDTA). Incubate for 5 min at 37 &#176;C. Then, add 4 mL of OP9 media and mechanically dissociate the cell layer by pipetting to make a single-cell suspension.</li>
	<li>Transfer the cell suspension into a 50 mL conical tube through a 100 &#956;m cell strainer to avoid cell clumps. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 12 mL of OP9 media.</li>
	<li>Add 8 mL of OP9 media to six new 10 cm cell culture Petri dishes. Plate 2 mL of OP9/DLL1 cell suspension from step 2.3 onto each new 10 cm dish. Rock the dish side-to-side and then front-to-back to ensure even distribution of OP9/DLL1 over the dish.</li>
	<li>Incubate at 37 &#176;C. Repeat passage every 2 - 3 days when cells reach confluency. 
	NOTE: It is important to make enough frozen stock of OP9/DLL1 cells and thaw a new stock every 4-6 weeks.</li>
</ol>
3. In Vitro Differentiation of hiPSCs into CD8&#945;&#946;+ Single Positive (SP) T Cells
<ol>
	<li>Prepare gelatinized OP9/DLL1 dishes one week before co-culture with hiPSCs. To prepare 0.1% gelatin solution, add 5 mL of room temperature (RT) tissue-grade stock gelatin solution to 500 mL of PBS.
	<ol>
		<li>Coat 3 new 10 cm cell culture Petri dishes by adding 4 mL per dish of 0.1% gelatin. Incubate 30 min at 37 &#176;C.</li>
		<li>Aspirate gelatin and add 8 mL of OP9 media to each dish. Passage one confluent dish of OP9/DLL1 (as done in section 2 above) to three gelatin pre-coated dishes.</li>
	</ol>
	</li>
	<li>After 4 days, add 10 mL of OP9 media to each 10 cm dish of OP9/DLL1 on gelatin for 20 mL of media per dish.</li>
	<li>After 7 - 8 days, begin the hiPSC co-culture on OP9/DLL1 confluent dishes (differentiation day 0).
	<ol>
		<li>Aspirate spent media from confluent 10 cm dish of hiPSCs on MEF. Add 10 mL of OP9 media. Cut and detach hiPSC colonies using a disposable cell passaging tool, as done in steps 1.2.3 and 1.2.4.</li>
		<li>Transfer 350 - 600 clumps of cut colonies onto a 10 cm pre-gelatinized OP9/DLL1 dish (step 3.1) with 10 mL of fresh OP9 media using a 200 &#956;L pipette. Rock the culture dish side-to-side and then front-to-back to ensure an even distribution of colonies. 
		NOTE: Alternatively, pre-formed hiPSC embryoid bodies (EBs) or small clump suspension may be used. However, using a disposable cell passaging tool or EB formation system is preferred to produce hiPSC clumps of uniform size.</li>
	</ol>
	</li>
	<li>On day 1, aspirate spent media and replace it with 20 mL of fresh OP9 media. hiPSC clumps co-cultured on OP9/DLL1 for 1 day will appear as small round monolayer colonies (Figure 1).</li>
	<li>On day 5, aspirate 10 mL of spent media and add 10 mL of fresh OP9 media. hiPSC colonies will begin to differentiate into primitive mesoderm, characterized by a multilayered dark center.</li>
	<li>On day 9, aspirate 10 mL of spent media and add 10 mL of fresh OP9 media. At this point, multilayered center structures will evolve into dome-like shapes, and a peripheral network-like area will become evident.</li>
	<li>On day 13, harvest hematopoietic progenitor cells (HPCs) (Figure 1). hiPSC-derived structures on day 13 are characterized by a dark central organoid surrounded by a network of dome-like areas, representative of hematopoietic zones (HZs) previously reported to enclose human embryonic stem cell-derived hematopoietic progenitors. 
	NOTE: The presence of the dome-like structures indicates a successful process even in the absence of dark centers. The inability to produce HPCs may be due to poor quality of OP9/DLL1, quality of FBS lot, confluency of iPSC clumps seeded onto OP9/DLL1 (350-600 clumps is optimal), and/or variations in potency of iPSC lines to produce hematopoietic precursors.
	<ol>
		<li>Aspirate spent media and wash 1x with 5 mL of 1x phenol red-free Hanks&#39; balanced salt solution modified with calcium and magnesium (HBSS).</li>
		<li>Aspirate HBSS and add 250 &#956;L of 5000 Units/mL collagenase IV in 10 mL of HBSS. Incubate at 37 &#176;C for 45 min. Aspirate HBSS with collagenase IV and wash once with 5 mL of PBS.</li>
		<li>Aspirate PBS and add 5 mL of 0.25% Trypsin-EDTA. Incubate at 37 &#176;C for 20 min. Then, add 4 mL of OP9 media and dissociate the cell layer by pipetting to make a single-cell suspension.</li>
		<li>Transfer the cell suspension into a 50 mL conical tube through a 100 &#956;m cell strainer. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 10 mL of OP9 media.</li>
		<li>Plate cell suspension onto a new gelatinized 10 cm cell-culture Petri dish (see steps 3.1.1 and 3.1.2). Incubate at 37 &#176;C for 45 min. Then, collect non-adherent cells by gentle pipetting.</li>
		<li>Transfer collected cell suspension into a 50 mL conical tube through a 100 &#956;m cell strainer. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 10 mL of differentiation media [OP9 media with 5 ng/mL human stem cell factor (hSCF), 5 ng/mL human Flt3 ligand (hFLT3L), and 5 ng/mL human interleukin 7 (hIL-7)].</li>
		<li>Plate the cell suspension onto a new 10 cm OP9/DLL1 confluent dish.</li>
	</ol>
	</li>
	<li>On day 16, passage the cells. 
	<ol>
		<li>Mechanically detach non-adherent cells by gentle pipetting and filter through a 100 &#956;m cell strainer. Centrifuge at 300 x g for 5 min at 4 &#176;C. Aspirate the supernatant and resuspend in 10 mL differentiation media.</li>
		<li>Plate the cell suspension onto a new 10 cm OP9/DLL1 confluent dish.</li>
	</ol>
	</li>
	<li>Continue passaging non-adherent cells every 5-7 days thereafter by repeating step 3.8.</li>
	<li>On day 35, enrich CD4+CD8+ double positive (DP) population and stimulate to produce CD8&#945;&#946;+ single positive (SP) T cells (Figure 2).
	<ol>
		<li>Mechanically detach non-adherent cells by gently pipetting and filtering through a 100 &#956;m cell strainer to remove clumps. Enrich the CD4+ cell population by CD4 magnetic bead isolation according to the manufacturer&#39;s protocol. 
		NOTE: The rationale for using CD4 magnetic beads is to remove CD4-CD8- double negative (DN) cells from the culture, as these have been demonstrated to cause direct killing of CD4+CD8+ DP cells after stimulation.</li>
		<li>Count live CD4 enriched cells using a Neubauer hemocytometer and Trypan blue dye. Suspend in OP9 media at a total concentration of 0.5 x 106 cells/mL. Aliquot 1 mL of the cell suspension (0.5 x 106 cells) into each well of a tissue culture flat bottom 24 healthy plates of confluent OP9/DLL1.</li>
		<li>Add 100 IU human interleukin 2 (hIL-2), 5 ng/mL hIL-7, 500 ng/mL anti-human CD3 antibody, and 2 &#956;g/mL anti-human CD28 antibody, then culture at 37 &#176;C.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 cm dish</td>
			<td>Corning, Inc.</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD3, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 561812, RRID:AB_1089628</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD34, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 348791, RRID:AB_400381</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD4, human</td>
			<td>Biolegend</td>
			<td>Cat# 344612, RRID:AB_2028479</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD43, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 560198, RRID:AB_1645460</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD7, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 555361, RRID:AB_395764</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8a, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 555369, RRID:AB_398595</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD8b, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 641057, RRID:AB_1645747</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-TCRb, human</td>
			<td>BD Biosciences</td>
			<td>Cat# 555548, RRID:AB_395932</td>
			<td></td>
		</tr>
		<tr>
			<td>CD28 human monoclonal antibody (15E8), pure functional grade</td>
			<td>Miltenyl Biotec</td>
			<td>130-093-375</td>
			<td></td>
		</tr>
		<tr>
			<td>CD3 human monoclonal antibody (OKT3), pure functional grade</td>
			<td>Miltenyl Biotec</td>
			<td>130-093-387</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 Microbeads, human</td>
			<td>Miltenyl Biotec</td>
			<td>130-045-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer 100 um</td>
			<td>Fisher Scientific</td>
			<td>22-363-549</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gemini</td>
			<td>100-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Flt-3 ligand</td>
			<td>R&#38;D Systems</td>
			<td>427-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin Solution 2%</td>
			<td>SIGMA-Aldritch</td>
			<td>G1393-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td>L-Glutamine supplement</td>
		</tr>
		<tr>
			<td>HBSS Mg+Ca+ Phenol-Red Free</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin-2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>Interleukin-7</td>
			<td>R&#38;D Systems</td>
			<td>407-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>iTAG MHC Tetramer HLA-A*0201 Mart1 Tetramer -ELAGIGILTV</td>
			<td>MBL</td>
			<td>Cat#TB-0009-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Mart1-hiPSC</td>
			<td>Vizcardo et al., Cell Stem Cell 2013</td>
			<td>RIKEN-IMS</td>
			<td></td>
		</tr>
		<tr>
			<td>Melan-A, MART 1 (26-35)</td>
			<td>InnoPep</td>
			<td>3146-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-Essential Amino Acids Solution</td>
			<td>Gibco</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;MEM powder</td>
			<td>Gibco</td>
			<td>61100061</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Embryonic Fibroblasts (MEF)</td>
			<td>Thermo Fisher Scientific</td>
			<td>C57BL/6 MEF MITC-TREATED 4M EACH; A34962</td>
			<td></td>
		</tr>
		<tr>
			<td>OP9/N-DLL1</td>
			<td>Riken Bioresource center</td>
			<td>Cat# RCB2927; RRID:CVCL_B220</td>
			<td>OP9/DLL1</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline pH 7.4 (1x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Primate ES Cell Medium</td>
			<td>Reprocell</td>
			<td>RCHEMD001</td>
			<td>Human ESC Culture Media</td>
		</tr>
		<tr>
			<td>Rhok inhibitor (Y-27632 dihydrochloride)</td>
			<td>Tocris</td>
			<td>1254</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Factor (SCF)</td>
			<td>R&#38;D Systems</td>
			<td>455-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>StemPro</td>
			<td>EZPassage</td>
			<td>23181-010</td>
			<td></td>
		</tr>
		<tr>
			<td>T2-tumor</td>
			<td>ATCC</td>
			<td>T2 (174 x CEM.T2) (ATCC&#174; CRL-1992&#8482;)</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200-072</td>
			<td></td>
		</tr>
		<tr>
			<td>U Bottom 96 well plate</td>
			<td>Corning, Inc.</td>
			<td>3799</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Good, M. L., et al. <a href="https://app.jove.com/v/59997">Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to generate tumor antigen-specific induced pluripotent stem cell (iPSC)-derived CD8&#945;&#946;+ T Cells. Tumor antigen-specific iPSCs are grown on a stromal cell monolayer and differentiated into hematopoietic progenitor cells (HPCs). The HPCs are differentiated into CD4+ CD8+ double-positive T cells using a differentiation medium. These cells are then stimulated to generate single-positive T cells expressing CD8.

<img alt="Figure 1" src="/files/ftp_upload/22280/22280_Figure1.jpg" /> 
Figure 1: Generation of hiPSC-derived hematopoietic progenitor cells. (A) Schematic overview of the differentiation of hiPSCs to hematopoietic lineage using OP9/DLL1 co-culture. (B) Appearance of hiPSC-derived structures on days 1 (top left), 3 (top right), 7 (bottom left), and 13 (bottom right). Scale bars = 100 &#181;m. (C) Flow cytometric analysis of hiPSC-derived CD34+CD43+ hematopoietic progenitor cells on day 13. Data represent six independent experiments (n = 1 to 2).
<img alt="Figure 2" src="/files/ftp_upload/22280/22280_Figure2.jpg" /> 
Figure 2: hiPSC differentiation into Mart1+ CD4+CD8&#945;&#946;+ DP T cells. (A) Schematic overview of the differentiation of hiPSC-derived hematopoietic lineage to immature T cells using OP9/DLL1 co-culture. (B) Flow cytometric analysis of CD4 vs. CD8&#945;, CD3 vs. CD8&#946;, and MART-1 tetramer expression in hiPSC-derived T cells on day 35. Gated on lymphocytes, single cells, PI negative. Data represent three independent experiments (n = 3 - 8).

1. Culturing Human iPSCs (hiPSCs) on Mouse Embryonic Fibroblasts (MEF)
NOTE: Alternative methods for culturing hiPSCs can also be used, including but not ...

Culture induced pluripotent stem cells on a feeder layer of stromal cells on a gelatin matrix. 
 The feeder layer mimics the natural extracellular environment, guiding stem cell differentiation into dome-shaped organoids containing hematopoietic progenitor cells. 
 Add enzymes to dissociate the co-cultured cells. 
 Transfer the cells to a fresh gelatin-coated dish for stromal cell adhesion. 
 Collect the non-adhered hematopoietic progenitor cells in a differentiation medium. 
 Culture them on a fresh feeder layer to promote progenitor cell differentiation into double-positive T cells with CD4 and CD8 receptors and double-negative T cells lacking both receptors. 
 Transfer the cells to a tube. Incubate them with magnetic beads carrying CD4-specific antibodies that selectively tag the double-positive T cells. 
 Use a magnetic separator to isolate the double-positive cells and culture them on a fresh feeder layer. 
 Supplement with a cytokine and antibody mix to stimulate the cells, inducing CD4 receptor downregulation and promoting their differentiation into CD8-single positive cells.

19 Views. Получение одиночных положительных CD8 Т-клеток из индуцированных плюрипотентных стволовых клеток

Video: Получение одиночных положительных CD8 Т-клеток из индуцированных плюрипотентных стволовых клеток - Experiment

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.

Эффективная дифференциация стволовых клеток человека в клетки печени

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

JoVE Journal

Developmental Biology

Scalable Generation of Mature Cerebellar Organoids from Human Pluripotent Stem Cells and Characterization by Immunostaining

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can trigger cerebellar dysfunction. Most of the current knowledge about disease-related neuronal phenotypes is based on postmortem tissues, which makes understanding of disease progression and development difficult. Animal models and immortalized cell lines have also been used as models for neurodegenerative disorders. However, they do not fully recapitulate human disease. Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling and provide a valuable source for regenerative approaches. In recent years, the generation of cerebral organoids from patient-derived iPSCs improved the prospects for neurodegenerative disease modeling. However, protocols that produce large numbers of organoids and a high yield of mature neurons in 3D culture systems are lacking. The protocol presented is a new approach for reproducible and scalable generation of human iPSC-derived organoids under chemically-defined conditions using scalable single-use bioreactors, in which organoids acquire cerebellar identity. The generated organoids are characterized by the expression of specific markers at both mRNA and protein level. The analysis of specific groups of proteins allows the detection of different cerebellar cell populations, whose localization is important for the evaluation of organoid structure. Organoid cryosectioning and further immunostaining of organoid slices are used to evaluate the presence of specific cerebellar cell populations and their spatial organization.

This protocol describes a dynamic culture system to produce controlled size aggregates of human pluripotent stem cells and further stimulate differentiation in cerebellar organoids under chemically-defined and feeder-free conditions using a single-use bioreactor.

Масштабируемое поколение зрелых мозжечковых органоидов из плюрипотентных стволовых клеток человека и характеристика иммуностимулированием

The cerebellum plays a critical role in the maintenance of balance and motor coordination, and a functional defect in different cerebellar neurons can ...

Bioengineering

Production and Characterization of Human Macrophages from Pluripotent Stem Cells

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key players in health and disease, acting as phagocytes during immune defense and mediating trophic, maintenance, and repair functions. Although it has been possible to study some of the molecular processes involved in human macrophage function, it has proved difficult to apply genetic engineering techniques to primary human macrophages. This has significantly hampered our ability to interrogate the complex genetic pathways involved in macrophage biology and to generate models for specific disease states. An off-the-shelf source of human macrophages that is amenable to the vast arsenal of genetic manipulation techniques would, therefore, provide a valuable tool in this field. We present an optimized protocol that allows for the generation of macrophages from human induced pluripotent stem cells (iPSCs) in vitro. These iPSC-derived macrophages (iPSC-DMs) express human macrophage cell surface markers, including CD45, 25F9, CD163, and CD169, and our live-cell imaging functional assay demonstrates that they exhibit robust phagocytic activity. Cultured iPSC-DMs can be activated to different&#160;macrophage states that display altered gene expression and phagocytic activity by the addition of LPS and IFNg, IL4, or IL10. Thus, this system provides a platform to generate human macrophages carrying genetic alterations that model specific human disease and a source of cells for drug screening or cell therapy to treat these diseases.

This protocol describes the robust generation of macrophages from human induced pluripotent stem cells, and methods for their subsequent characterization. Cell surface marker expression, gene expression, and functional assays are used to assess the phenotype and function of these iPSC-derived macrophages.

Производство и характеристика макрофагов человека из плюрипотентных стволовых клеток

Macrophages are present in most vertebrate tissues and comprise widely dispersed and heterogeneous cell populations with different functions. They are key ...

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells

ТРАНСКРИПТ

Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells