We are extremely grateful to all members of the Mao and Hu Lab, past and present, for the interesting discussions and great contributions to the project. We are thankful to the National Clinical Research Center for Child Health for the great support. This study was financially supported by the National Natural Science Foundation of China (U20A20351 to Jianhua Mao, 82200784 to Lidan Hu), the Natural Science Foundation of Zhejiang Province of China (No. LQ22C070004 to Lidan Hu).

1. hiPSCs maintenance
<ol>
	<li>Thawing of hiPSC
	<ol>
		<li>Take out the cells from the liquid nitrogen and quickly thaw cells in a 37 &#176;C water bath. Transfer the thawing cells to a 15 mL tube prepared with 3 mL of iPSC maintenance medium (Table of Materials). Gently mix the medium.</li>
		<li>Centrifuge at 300 x g for 5 min. Remove the supernatant, and gently resuspend the cells in 1 mL of iPSC maintenance medium with 10 &#181;M Y-27632 (pipette the cells up and down 2-3 times).</li>
		<li>Transfer the cells suspension into a 6-well tissue culture plate coated with growth factor reduced (GFR)-extracellular matrix gel (1:100) and 2 mL of iPSC maintenance medium with 10 &#181;M Y-27632 added in advance (about 4 x 104 cells/cm2).</li>
		<li>Culture the cells for 5-6 days (80%-90% confluence) at 37 &#176;C, 5% CO2 with iPSC maintenance media change every day.</li>
	</ol>
	</li>
	<li>Passage of hiPSC
	<ol>
		<li>Remove the iPSC maintenance medium from the 6-well plate. Wash the hiPSCs once with DPBS.</li>
		<li>Add 700-800 &#181;L of 0.48 mM EDTA solution and incubate for 1 min at room temperature (RT), then remove the digestion solution. Continue to incubate at 37 &#176;C temperature for 3-5 min.</li>
		<li>When cells are digested into sheets (do not digest cells into single cells), add 1 mL of iPSC maintenance medium with 10 &#181;M Y-27632 to terminate digestion. Carefully pipette the cells up and down 2-3 times.</li>
		<li>Transfer the cell suspension into a 6-well tissue culture plate coated with growth factor reduced (GFR)-extracellular matrix gel (1:100) and 2 mL of iPSC maintenance medium with 10 &#181;M Y-27632 added in advance. 
		NOTE: The passaging ratio ranges from 1:6 to 1:20 (about 4 x 104 cells/cm2), and the mean aggregation size is approximately 50-200 &#181;m.</li>
		<li>Culture the cells until 80%-90% confluency (5-6&#160;days) at 37 &#176;C, 5% CO2 with iPSC maintenance media change every day.</li>
	</ol>
	</li>
</ol>
2. MSCs differentiation from hiPSCs via EB formation
NOTE: The method is derived from previous literature21,22,23,24. An overview of the method is illustrated in Figure 1. The characteristics of the method are summarized in Table 1.
<ol>
	<li>Preparation of the medium
	<ol>
		<li>Prepare MSC differentiation medium by supplementing &#945;-MEM with 1% (v/v) GlutaMAX, 10% (v/v) FBS, 10 ng/mL FGF2, and 5 ng/mL TGF&#946;.</li>
		<li>Prepare MSC maintenance medium by supplementing &#945;-MEM with 1% (v/v) GlutMAX, 10% (v/v) FBS, and 1 ng/mL FGF2.</li>
	</ol>
	</li>
	<li>Preparation of hiPSCs
	<ol>
		<li>Culture hiPSCs lines (passage at least 2-3 times after thawing) in iPSC maintenance medium on a 6-well tissue culture plate coated with growth factor reduced (GFR)-extracellular matrix gel (1:100) at 37 &#176;C, 5% CO2 until 80%-90% confluency.</li>
	</ol>
	</li>
	<li>Day 0: EB formation
	<ol>
		<li>Remove the iPSC maintenance medium from the 6-well plate. Wash the hiPSCs once with DPBS.</li>
		<li>Add 700-800 &#181;L of 0.48 mM EDTA solution and incubate for 1 min at RT, then remove the digestion solution. Continue to incubate at 37 &#176;C temperature for 3-5 min.</li>
		<li>When cells are digested into sheets (do not digest cells into single cells), add 2 mL of iPSCs maintenance medium (containing 10 &#181;M Rock inhibitor and 1:100 GFR- extracellular matrix gel) to terminate digestion.</li>
		<li>Transfer the cells to a 6-well low attachment plate. Seed the cells in a ratio of 2 wells of tissue culture plate to 1 well of low attachment plate. Incubate the cells in a shaker (60 rpm/min) at 37 &#176;C, 5% CO2 for 24 h to form spherical EBs.</li>
	</ol>
	</li>
	<li>Day 1-Day 7: EB differentiation
	<ol>
		<li>Transfer the EBs to the centrifuge tube with a Pasteur pipette (without destroying EBs) and let the EBs naturally sediment for 5-10 min at RT. Then, remove the supernatant.</li>
		<li>Transfer the EBs to a 6-well low attachment plate with 2 mL of MSC differentiation medium. Culture the EBs on the shaker at 37 &#176;C, 5% CO2 for 7 days with medium change once.</li>
	</ol>
	</li>
	<li>Day 8-Day 17: EB inoculation
	<ol>
		<li>Transfer the EBs to the centrifuge tube with a Pasteur pipette (without destroying EBs) and let the EBs naturally sediment for 5-10 min. Then, remove the supernatant.</li>
		<li>Transfer the EBs to a GFR-extracellular matrix gel-coated 6-well plate with 2 mL of MSC maintenance medium. Culture until 90% confluency (~10 days) with regular media change after cell adhesion.</li>
	</ol>
	</li>
	<li>Day 18-Day 27: MSCs maturation and expansion
	<ol>
		<li>Treat the EB-derived culture with a dissociation solution at 37 &#176;C for 5-10 min (digestion time varies due to the presence of different cell types).</li>
		<li>When part of the cells are digested into single cells, add 2 mL of the MSC maintenance medium to terminate digestion and transfer the cell suspension to a centrifuge tube. Wash the remaining undigested cells once with DPBS and digest again. Repeat several times until all cells are digested into single cells. Centrifuge the cell suspension at 250 x g for 5 min, then remove the supernatant.</li>
		<li>Seed the cells on a Gelatin-coated culture plate (about 2 x 105 cells/cm2). Culture until 90% confluency in MSC maintenance medium with medium change regularly. Designate this generation of cells as passage 0 (P0) at this point.</li>
		<li>In order to make MSC pure&#160;and mature, continue to passage the cells twice at a 1:3 split ratio in approximately 6 days. Most of the cells present fibroblast-like morphology (spindle-shaped).</li>
	</ol>
	</li>
</ol>
3. MSCs differentiation from hiPSCs via monolayer culture
NOTE: The method is derived from previous literature25,26,27,28. An overview of this method is illustrated in Figure 1. The characteristics of the method are summarized in Table 1.
<ol>
	<li>Preparation of the medium
	<ol>
		<li>Prepare the MSC maintenance medium by supplementing &#945;-MEM with 1% (v/v) GlutMAX, 10% (v/v) FBS, and 1 ng/mL FGF2.</li>
	</ol>
	</li>
	<li>Preparation of hiPSCs
	<ol>
		<li>Culture hiPSCs lines (passage at least 2-3 times after thawing) in the iPSC maintenance medium on a 6-well tissue culture plate coated with growth factor reduced (GFR)-extracellular matrix gel (1:100) at 37 &#176;C, 5% CO2 until 50%-60% confluency.</li>
	</ol>
	</li>
	<li>Day 0-Day 13: Differentiation by direct monolayer cultures
	<ol>
		<li>Remove the iPSC maintenance medium from the 6-well plate.</li>
		<li>Directly add 2 mL of the MSC maintenance medium and culture for 14 days at 37 &#176;C, 5% CO2, with the media change every day.</li>
	</ol>
	</li>
	<li>Day 14-Day 35: MSCs maturation by repeated passage
	<ol>
		<li>Treat the monolayer cultures with a dissociation solution at 37 &#176;C for 5-10 min (digestion time varies due to the presence of different cell types).</li>
		<li>When the cells are digested into single cells, add 2 mL of MSC maintenance medium to terminate digestion and transfer the cell suspension to centrifuge tube. Wash the remaining undigested cells once with DPBS and digest again. Repeat several times until all cells are digested into single cells. Centrifuge the cell suspension at 250 x g for 5 min, then remove the supernatant.</li>
		<li>Seed the cells on a gelatin-coated culture dish (about 2 x 105 cells/cm2). Culture until 90% confluency in MSC maintenance medium with media change regularly. Designate this generation of cells as passage 0 (P0) at this point.</li>
		<li>In order to make MSC pure&#160;and mature, continue to passage the cells 6 times at a 1:3 split ratio in approximately 18 days. Most of the cells present fibroblast-like morphology (spindle-shaped).</li>
	</ol>
	</li>
</ol>
4. Surface antigens analysis of hiPSC-driving MSCs by flow cytometry
NOTE: Similar to the surface antigens of bone marrow-derived MSCs, hiPSCs-driving MSC express CD105, CD73 and CD90, but do not express CD45, CD3429. In addition, hiPSCs can be used as negative control cells. Surface antigens analysis of hiPSC-driving MSCs and hiPSCs by flow cytometry are shown in Figure 2.
<ol>
	<li>Treat the hiPSC-driving MSCs with a dissociation solution at 37 &#176;C for 2-3 min. Treat the hiPSCs with 0.48 mM EDTA dissociation reagent and incubate for 1 min at RT, then remove the dissociation reagent. Continue to incubate at 37 &#176;C temperature for 3-5 min.</li>
	<li>When the cells are digested into single cells, add medium (MSC maintenance medium to MSCs and iPSC maintenance medium to iPSCs) to terminate digestion.</li>
	<li>Centrifuge the cell suspension at 350 x g for 5 min, then remove the supernatant.</li>
	<li>Wash the cells once with cold DPBS, centrifuge at 350 x g for 5 min, and then remove the supernatant.</li>
	<li>Count and resuspend the cells in cold 10% FBS-DPBS (v/v) solution at 10 x 106 cells/mL and distribute 100 &#181;L/tube of cell suspension (1 x 106 cells/tube) into 1.5 mL centrifuge tubes.</li>
	<li>Add 5 &#181;L of human Fc receptor-blocking solution to 100 &#181;L of cell suspension. Incubate for 5-10 min at RT to perform nonspecific blocking.</li>
	<li>Centrifuge at 350 x g for 5 min and remove the supernatant. Wash the cells twice with a cold 2% FBS-DPBS (v/v) solution.</li>
	<li>Resuspend the cells in cold 100 &#181;L of 2% FBS-DPBS (v/v) solution.</li>
	<li>Add 5 &#181;L (1 test) of anti-CD34-FITC and 5 &#181;L (1 test) of anti-CD45-APC to the 100 &#181;L cell 
	Suspension. Add 5 &#181;L (1 test) of anti-CD73-APC, 5 &#181;L (1 test) of anti-CD90-FITC and 5 &#181;L (1 test) of anti-CD105-PE to another 100 &#181;L cell suspension tube. Add 5 &#181;L of human IgG1 isotype control FITC, human IgG1 isotype control PE and human IgG1 isotype control APC to the third 100 &#181;L cell suspension tube. Incubate for 15-20 min on ice in the dark. Meanwhile, prepare single-staining beads.</li>
	<li>Wash the cells twice with a cold 2% FBS-DPBS (v/v) solution.</li>
	<li>Add 300 &#181;L of cold 2% FBS-DPBS (v/v) solution to resuspend the cells, and filter via 200 mesh screen filter.</li>
	<li>Analyze the samples using flow cytometry. Analyze the cell suspension stained with isotype control antibodies and set gate. Then analyze cell suspension stained with target antibodies.</li>
</ol>
5. Osteogenic differentiation of hiPSC-driving MSCs
NOTE: The hiPSC-driving MSCs possess osteogenic differentiation potential (Figure 3A, B). The protocol for osteogenic differentiation is given below.
<ol>
	<li>Prepare osteogenic induction medium by supplementing &#945;-MEM with 10% FBS, 100 nM dexamethasone, 10 mM beta-glycerophosphate, and 100 &#181;M ascorbic acid.</li>
	<li>Seed 1 x 105 MSCs into a gelatin-coated 48-well plate and culture until it is 60%-70% confluent.</li>
	<li>Remove the medium and add 0.3 mL of osteogenic induction medium. Culture the cells in an osteogenic induction medium for 14 days at 37 &#176;C, 5% CO2, with media change every 3 days.</li>
	<li>Remove the medium and wash with DPBS once.</li>
	<li>Add 0.3 mL of 4% PFA and incubate at RT for 30 min.</li>
	<li>Remove PFA. Rinse the cells with 0.3 mL of DPBS for 10 min at RT and repeat 3 times.</li>
	<li>Remove the DPBS, add 0.2 mL of Alizarin red staining solution, and incubate for 30 min at RT.</li>
	<li>Remove the staining solution, rinse the cells with 0.3 mL DPBS for 10 min at RT, and repeat 3 times.</li>
	<li>Observe the staining of calcium deposition under a light microscope.</li>
</ol>
6. Adipogenic differentiation of hiPSC-driving MSCs
NOTE: The hiPSC-driving MSCs possess adipogenic differentiation potential (Figure 3C, D). The protocol for adipogenic differentiation is given below.
<ol>
	<li>Preparare adipogenic induction medium by supplementing &#945;-MEM with 10% FBS, 1 &#181;M dexamethasone, 1 &#181;M IBMX, 10 &#181;g/mL insulin, and 100 &#181;M indomethacin. Preparare adipogenic maintenance medium by supplementing &#945;-MEM with 10% FBS, and 10 &#181;g/mL insulin.</li>
	<li>Seed 1 x 105 MSCs into a gelatin-coated 48-well plate and culture until the cell density reaches 90%.</li>
	<li>Remove the medium and add 0.3 mL of adipogenic induction medium. Continue to culture the cells in the adipogenic induction medium for 4 days at 37 &#176;C, 5% CO2.</li>
	<li>Remove the medium and add 0.3 mL of adipogenic maintenance medium. Continue to culture the cells in the adipogenic maintenance medium for 3 days at 37 &#176;C, 5% CO2.</li>
	<li>Repeat steps 6.3 and 6.4 2-3 times until adipocyte differentiation and maturation.</li>
	<li>Remove the medium and wash with DPBS once.</li>
	<li>Add 0.3 mL of 4% PFA and incubate at RT for 10 min. Remove the PFA and rinse 2 times with DPBS.</li>
	<li>Apply Oli Red O stain per the manufacturer&#39;s instruction and observe the lipid droplets under a light microscope.</li>
</ol>
7. Chondrogenic differentiation of hiPSC-driving MSCs
NOTE: The hiPSC-driving MSCs possess chondrogenic differentiation potential (Figure 3E, F). The protocol for chondrogenic differentiation is given below.
<ol>
	<li>Prepare chondroblast induction medium by supplementing &#945;-MEM with 10% FBS, 100 nM dexamethasone, 1% Insulin-Transferrin-Selenium (ITS), 10 &#181;M ascorbic acid, 1 mM sodium pyruvate, 50 &#181;g/mL proline, 0.02 nM transforming growth factor &#946;3 (TGF&#946;3), and 0.5 &#181;g/mL bone morphogenetic protein 6 (BMP-6).</li>
	<li>Collect 2.5 x 105 hiPSC-driving MSCs and centrifuge the cells at 150 x g for 5 min, then remove the supernatant. Suspend the cells in 1 mL of &#945;-MEM and then centrifuge at 150 x g for 5 min.</li>
	<li>Remove the supernatant and suspend the cells in 0.5 mL of chondrogenic induction medium. Centrifuge the cells at 150 x g for 5 min.</li>
	<li>Directly unscrew the lid and culture for 21 days with chondrogenic induction media change every 3 days at 37 &#176;C, 5% CO2. Flick the centrifuge tube gently to suspend the chondrogenic pellets&#160;(without destroying chondrogenic pellets) every day during chondrogenic differentiation.</li>
	<li>Remove the supernatant and wash with 0.5 mL of PBS twice.</li>
	<li>Add 0.3 mL of 4% PFA and fix for 24 h.</li>
	<li>Paraffin embed the chondrogenic pellets, and then section them (3 &#181;m). Stain the sections in toluidine blue (1%) for 30 min.</li>
	<li>Observe the extracellular chondrocyte matrix under a light microscope.</li>
</ol>

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

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W., 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=Exosomes+secreted+by+human-induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells+attenuate+limb+ischemia+by+promoting+angiogenesis+in+mice.">Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.</a> Stem Cell Research &amp; Therapy. 6 (1), 10 (2015).</li><li>Kang, R., 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=Mesenchymal+stem+cells+derived+from+human+induced+pluripotent+stem+cells+retain+adequate+osteogenicity+and+chondrogenicity+but+less+adipogenicity.">Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.</a> Stem Cell Research &amp; Therapy. 6 (1), 144 (2015).</li><li>Wang, L. T., 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=Differentiation+of+mesenchymal+stem+cells+from+human+induced+pluripotent+stem+cells+results+in+downregulation+of+c-Myc+and+DNA+replication+pathways+with+immunomodulation+toward+CD4+and+CD8+cells.">Differentiation of mesenchymal stem cells from human induced pluripotent stem cells results in downregulation of c-Myc and DNA replication pathways with immunomodulation toward CD4 and CD8 cells.</a> Stem Cells. 36 (6), 903-914 (2018).</li><li>Dominici, M., 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Kim, S., Kim, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem-like+cells+for+producing+extracellular+vesicles.">Generation of mesenchymal stem-like cells for producing extracellular vesicles.</a> World Journal of Stem Cells. 11 (5), 270-280 (2019).</li></ol>

The authors have nothing to disclose.

In this protocol, two representative methods of differentiating hiPSCs into MSCs were examined20,21,22,23,24,25,26,27,28,30. Both methods were capable of derivating MSCs from hiPSCs. The ...

Mesenchymal stromal cells (MSCs) are mesoderm-derived adult pluripotent stem cells1. MSCs are present in almost all connective tissues2. Since MSCs were first discovered in the 1970s and successfully isolated from bone marrow in 1987 by Friedenstein et al.3,4,5, a variety of human somatic (including fetal and adult) tissues have been used for isolating MSCs such as bone, cartilage, tendon, muscle, adipose tissue, and hematopoietic-supporting stroma1,2,6,7. MSCs demonstrate high proliferative capabilities and plasticity to differentiate into many somatic cell lineages and could migrate to injured and inflamed tissues2,8,9. These properties make MSCs a potential candidate for regenerative medicine10. However, somatic tissue-derived MSCs (st-MSCs) are restricted by limited donation, limited cell proliferative capacity, quality variations, and biosafety concern for possible transmission of pathogens, if any, from the donors11,12.
Human induced pluripotent stem cells (hiPSCs) are derived from adult cells reprogramming with transcription factors (Oct4, Sox2, Klf4, and c-Myc), which have similar functions as embryonic stem cells13,14. They can self-renew and possess the potential of differentiating into any type of somatic cells, including MSCs. Compared with st-MSCs, iPSC-MSCs has the advantage of unlimited supply, lower cost, higher purity, convenience in quality control, easy for scale production and gene modification15,16,17.
Due to these advantages of iPSC-MSCs, a variety of methods driving MSC from iPSC have been reported. These differentiation methods have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer cultures11,18,19,20. Herein, a representative approach for each of the two methodologies was characterized. Furthermore, comparisons between two representative approaches based on time, cost, proliferative ability, expression of MSC biomarkers, and differentiation capability in vitro were also accessed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alizarin red staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0148S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD105 (PE)</td>
			<td>Biolegend</td>
			<td>323206</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD34 (FITC)</td>
			<td>Biolegend</td>
			<td>343503</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD45 (APC)</td>
			<td>Biolegend</td>
			<td>304011</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD73( APC)</td>
			<td>Biolegend</td>
			<td>344006</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human-CD90 (FITC)</td>
			<td>Biolegend</td>
			<td>328108</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Solarbio</td>
			<td>A8100</td>
			<td></td>
		</tr>
		<tr>
			<td>BMP-6</td>
			<td>Novoprotein</td>
			<td>C012</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon dioxide level shaker</td>
			<td>Crystal</td>
			<td>CO-06UC6</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation Beads</td>
			<td>BioLegend</td>
			<td>424601</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>STEMCELL Technology</td>
			<td>07959</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1254</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12&#160; medium</td>
			<td>Servicebio</td>
			<td>G4610</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HAKATA</td>
			<td>HS-FBS-500</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Stemcell</td>
			<td>78003.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G2500-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control APC</td>
			<td>BioLegend</td>
			<td>403505</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control FITC</td>
			<td>BioLegend</td>
			<td>403507</td>
			<td></td>
		</tr>
		<tr>
			<td>human IgG1 isotype control PE</td>
			<td>BioLegend</td>
			<td>403503</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TGF-&#946;1</td>
			<td>Stemcell</td>
			<td>78067</td>
			<td></td>
		</tr>
		<tr>
			<td>Human TruStain FcX&#160;</td>
			<td>BioLegend</td>
			<td>422301</td>
			<td></td>
		</tr>
		<tr>
			<td>IBMX</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>ST1398</td>
			<td></td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Solarbio</td>
			<td>SI9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>P3376</td>
			<td></td>
		</tr>
		<tr>
			<td>iPSC maintenance medium</td>
			<td>STEMCELL Technology</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Media Supplement</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0341-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, growth factor reduced</td>
			<td>BD Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Oli Red O staining kit</td>
			<td>Beyotime&#160;Biotechnology</td>
			<td>C0158S</td>
			<td></td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>Solarbio</td>
			<td>P0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>ThermoFisher</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF&#946;3</td>
			<td>Novoprotein</td>
			<td>CJ44</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue staining kit</td>
			<td>Solarbio</td>
			<td>G2543</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme(1x)&#160;</td>
			<td>Gibco</td>
			<td>12604013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Low Attachment 6 Well Plate</td>
			<td>Costar</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Versene</td>
			<td>Gibco</td>
			<td>15040-66</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Stemcell</td>
			<td>72304</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-MEM</td>
			<td>Hyclone</td>
			<td>SH30265</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Solarbio</td>
			<td>G8100</td>
			<td></td>
		</tr>
	</tbody>
</table>

comparison of two representative methods for differentiation of human induced pluripotent stem cells into mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Following the protocol (Figure 1A), hiPSCs were differentiated into MSCs via the EB formation and monolayer culture methods. During differentiation, the cells showed different representative morphologies (Figure 1B,C).
As shown in Figure 1B, the hiPSCs colonies display typical compact morphology before differentiation with a clear border composed of tightly packed cells. Uniform spherical...

Watch this Scientific Journal Video about Advancements in iPSCs and Genetic Disease Research at JoVE.com

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

The scope of our study is a mechanism, and a therapeutic strategy research of genetic disease. Our research is based on multilevel disease models, such as cell lines, iPSCs, organoids, and mice. iPSCs have a distinct advantage over other models.iPSCs from patients, and any subsequent derived cells and organoids share the same genome with the patients, which provides a bank of patient cells and models for many disease. Also, with the methods of established iPSCs from patients are quite mature. Many challenges remain in the direction of differentiating of iPSCs into specific cells and organoids, with a deepening understanding of development mechanisms in current development will occur in the field.

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795 Views.  The Children's Hospital, Zhejiang University School of Medicine. The scope of our study is a mechanism, and a therapeutic strategy research of genetic disease. Our research is based on multilevel disease models, such as cell lines, iPSCs, organoids, and mice. iPSCs have a distinct advantage over other models.iPSCs from patients, and any subsequent derived cells and organoids share the same genome with the patients, which provides a bank of patient cells and models for many disease. Also, with the methods of established iPSCs from patients are quite ...

Video: Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

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

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...

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

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

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

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

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

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

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

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

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Maintenance of Primate Induced Pluripotent Stem Cells Derived from Urine

Cross-species approaches studying primate pluripotent stem cells and their derivatives are crucial to better understand the molecular and cellular mechanisms of disease, development, and evolution. To make primate induced pluripotent stem cells (iPSCs) more accessible, this paper presents a non-invasive method to generate human and non-human primate iPSCs from urine-derived cells, and their maintenance using a feeder-free culturing method.
The urine can be sampled from a non-sterile environment (e.g., the cage of the animal) and treated with a broad-spectrum antibiotic cocktail during primary cell culture to reduce contamination efficiently. After propagation of the urine-derived cells, iPSCs are generated by a modified transduction method of a commercially available Sendai virus vector system. First&#160;iPSC colonies may already be visible after 5 days, and can be picked after 10 days at the earliest. Routine clump passaging with enzyme-free dissociation buffer supports pluripotency of the generated iPSCs for more than 50 passages.

The present protocol describes a method to isolate, expand, and reprogram human and non-human primate urine-derived cells to induced pluripotent stem cells (iPSCs), as well as instructions for feeder-free maintenance of the newly generated iPSCs.