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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<li>Hu, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exosomes+secreted+by+human-induced+pluripotent+stem+cell-derived+mesenchymal+stem+cells+attenuate+limb+ischemia+by+promoting+angiogenesis+in+mice%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice.</a> Stem Cell Research & Therapy. 6 (1), 10(2015).</li>
<li>Kang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+stem+cells+derived+from+human+induced+pluripotent+stem+cells+retain+adequate+osteogenicity+and+chondrogenicity+but+less+adipogenicity%5BTitle%5D)+AND+%22Stem+Cell+Research+%26+Therapy%22%5BJournal%5D)">Mesenchymal stem cells derived from human induced pluripotent stem cells retain adequate osteogenicity and chondrogenicity but less adipogenicity.</a> Stem Cell Research & Therapy. 6 (1), 144(2015).</li>
<li>Wang, L. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?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%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">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/pubmed?term=((Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">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/pubmed?term=((Generation+of+mesenchymal+stem-like+cells+for+producing+extracellular+vesicles%5BTitle%5D)+AND+%22World+Journal+of+Stem+Cells%22%5BJournal%5D)">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><tbody><tr><td>Alizarin red staining kit</td><td>Beyotime&amp;nbsp;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&amp;nbsp;Biotechnology</td><td>ST1254</td><td></td></tr><tr><td>DMEM/F12&amp;nbsp; 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-&amp;beta;1</td><td>Stemcell</td><td>78067</td><td></td></tr><tr><td>Human TruStain FcX&amp;nbsp;</td><td>BioLegend</td><td>422301</td><td></td></tr><tr><td>IBMX</td><td>Beyotime&amp;nbsp;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&amp;nbsp;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&amp;nbsp;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&amp;nbsp;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&amp;beta;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)&amp;nbsp;</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>&amp;alpha;-MEM</td><td>Hyclone</td><td>SH30265</td><td></td></tr><tr><td>&amp;beta;-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 ...

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Research

JoVE Journal

Biology

1.1K 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.

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

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Developmental Biology

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

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

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

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

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

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

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Robust Differentiation of Human iPSCs into a Pure Population of Adipocytes to Study Adipocyte-Associated Disorders

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the current differentiation methods have low efficiency and do not produce a homogenous population of adipocytes. Here, we circumvent this problem by using an all-trans retinoic-based method to produce mesenchymal stem cells (MSCs) in high yield. By regulating pathways governing cell proliferation, survival, and adhesion, our differentiation strategy allows the efficient generation of embryonic bodies (EBs) that differentiate into a pure population of multipotent MSCs. The high number of MSCs generated by this method provides an ideal source for generating adipocytes. However, sample heterogeneity resulting from adipocyte differentiation remains a challenge. Therefore, we used a Nile red-based method for purifying lipid-bearing mature adipocytes using FACS. This sorting strategy allowed us to establish a reliable way to model adipocyte-associated metabolic disorders using a pool of adipocytes with reduced sample heterogeneity and enhanced cell functionality.

The protocol allows the generation of a pure adipocyte population from induced pluripotent stem cells (iPSCs). Retinoic acid is used to differentiate iPSCs into mesenchymal stem cells (MSCs) which are used for producing adipocytes. Then, a sorting approach based on Nile red staining is used to obtain pure adipocytes.

Recent advances in induced pluripotent stem cell (iPSC) technology have allowed the generation of different cell types, including adipocytes. However, the ...

Differentiation and Characterization of Osteoclasts from Human Induced Pluripotent Stem Cells

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a single-cell suspension for further use in embryoid body induction. Following mesodermal induction, embryoid bodies undergo hematopoietic differentiation, producing a floating hematopoietic cell population. Subsequently, the harvested hematopoietic cells undergo a macrophage colony-stimulating factor maturation step and, finally, osteoclast differentiation. After osteoclast differentiation, osteoclasts are characterized by staining for TRAP in conjunction with a methyl green nuclear stain. Osteoclasts are observed as multinucleated, TRAP+ polykaryons. Their identification can be further supported by Cathepsin K staining. Bone and mineral resorption assays allow for functional characterization, confirming the identity of bona fide osteoclasts. This protocol demonstrates a robust and versatile method to differentiate human osteoclasts from iPSCs and allows for easy adoption in applications requiring large quantities of functional human osteoclasts. Applications in the areas of bone research, cancer research, tissue engineering, and endoprosthesis research could be envisioned.

This protocol presents the differentiation of human osteoclasts from induced pluripotent stem cells (iPSCs) and describes methods for the characterization of osteoclasts and osteoclast precursors.

This protocol details the propagation and passaging of human iPSCs and their differentiation into osteoclasts. First, iPSCs are dissociated into a ...

High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle &#8220;in a dish&#8221; for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.

The article describes the detailed methodology to efficiently differentiate human pluripotent stem cells into cardiomyocytes by selectively modulating the Wnt pathway, followed by flow cytometry analysis of reference markers to assess homogeneity and identity of the population.

There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Neuroscience

Monitoring Pluripotent Stem Cell Differentiation by Image-Based ML Strategy

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available PSC-to-functional cell differentiation systems are impeded by problems of severe line-to-line and batch-to-batch variability. Precise control of cell differentiation in real time is therefore important. In this protocol, we describe a non-invasive and intelligent strategy that overcomes the variability in cell differentiation by using bright-field image-based machine learning. Taking PSC-to-cardiomyocyte differentiation as an example, this methodology provides detailed information for control of the initial PSC state, early assessment and intervention in differentiation conditions, and elimination of the misdifferentiated cell contamination, together realizing consistently high-quality differentiation from PSCs to functional cells. In principle, this strategy can be extended to other cell differentiation or reprogramming systems with multiple steps to support cell manufacturing, as well as to further our understanding of the mechanisms during cell fate conversion.

Author Spotlight: Enhancing PSC-to-Functional Cell Differentiation Using ML Models Based on Live-Cell Bright-Field Imaging

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...

Differentiating Human-Induced Pluripotent Stem Cells into Microglia-Like Cells

Source: Funes, S. et al., <a href="https://app.jove.com/v/64323">Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes.</a>&#160;J. Vis. Exp. (2022)
This video demonstrates the procedure of the differentiation of human pluripotent cells into microglia-like cells. The process involves forming embryoid bodies, transferring them to coated wells, and using specific growth factors to guide differentiation into primitive macrophage precursors, which finally differentiate into microglia-like cells.

1. Microglia differentiation
NOTE: An overview of the protocol is summarized in&#160;Figure 1.

	Induced pluripotent stem cell (iPSC) culture
	NOTE: ...

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

Summary

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