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.

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

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

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.

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

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.

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

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Biology

MSCs Differentiation from hiPSCs via EB Formation and Monolayer Culture

To begin, culture hiPSCs in IPSC maintenance medium on a six-well tissue culture plate coated with growth factor reduced extracellular matrix gel. When the cells attain 80 to 90%v, remove the medium from the plate and wash the cells once with DPBS. Then add 700 to 800 microliters of 0.48 millimolar EDTA and incubate for one minute at room temperature.Remove the digestion solution and incubate the plate at 37 degrees Celsius for three to five minutes. When cells are digested into sheets, add two milliliters of IPSCs maintenance medium to terminate digestion. Transfer the cell suspension to a six-well low attachment plate.Incubate the cells at 37 degrees Celsius with 5%carbon dioxide and 60 RPM to form spherical embryo bodies or EBs. After 24 hours, using a past pipette, transfer the EBs to the centrifuge tube and allow it to sediment for five to 10 minutes before removing the supernatant. Add MSC differentiation medium to the tube and transfer the EBs to a six-well low attachment blade with two milliliters of MSCs differentiation medium.Culture the EBs on the shaker at 37 degrees Celsius with 5%carbon dioxide for seven days. On the eighth day, transfer the EBs to the centrifuge tube as demonstrated previously. Once the EBs are sedimented, remove the supernatant from the tube.Add MSC maintenance medium into the tube and transfer the EBs to a growth factor reduced extracellular matrix gel coated six-well plate with two milliliters of MSC maintenance medium. After cell adhesion, change the medium until the culture attains 90%confluency. Next, treat the EB derived culture with a dissociation solution at 37 degrees Celsius.When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium to terminate digestion and transfer the cell suspension to a centrifuge tube. Wash the remaining undigested cells from the wells once with DPBS. And digest again as demonstrated previously.Then centrifuge the cell suspension at 250g for five minutes. Remove the supernatant and seed the cells in a gelatin coated culture plate. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.Culture hiIPCs lines as demonstrated previously. When the cells attain 50 to 60%confluency, remove the IPSC maintenance medium from the plate. Add two milliliters of MSC maintenance medium.And culture for 14 days at 37 degrees Celsius and 5%carbon dioxide with daily medium change. For the maturation of MSCs, treat the monolayer culture with a dissociation solution at 37 degrees Celsius. When part of the cells are digested into single cells, add two milliliters of the MSC maintenance medium into the wells to terminate digestion and transfer the cell suspension to a centrifuge tube.Wash the remaining undigested cells once with DPBS and digest them as demonstrated previously. Then centrifuge the cell suspension as demonstrated previously and seed the cells on a gelatin coated culture dish. Culture the cells in MSC maintenance medium to 90%confluency with regular medium change.In the EB formation method, hiIPCs colonies exhibited a compact morphology before differentiation. After dissociation, uniform spherical EBs formed, which drew in volume over time. Subsequently, EBs transformed into adherent monolayer cells achieving 90%confluency by day 18 and acquiring a spindle shape morphology after two passages.Similarly, in the monolayer method, cells proliferated forming multilayer adherent cells. After packaging multiple times, cells matured into MSC with a typical spindle shape and swirling colony. Flow cytometry analysis revealed that hiIPCs were positive for CD90 and negative for CD34, CD45, CD105, and CD73.After differentiation, the hiIPCs driving MSCs expressed CD90, CD73, and CD105, while remaining negative for CD34 and CD45. Monolayer derived MSCs showed higher calcium deposit formation than the EB method. However, no significant differences were observed in adipogenic and chondrogenic differentiation abilities.The proliferation abilities of both methods were maintained for up to 20 passages.

773 Views.  The Children's Hospital, Zhejiang University School of Medicine.  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.

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

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