We thank M. Haraguchi and S. Tamaki for technical support and laboratory management and K. Shiroshita for taking photographs. HK was supported in part by the KAKENHI Grant from MEXT/JSPS (grant no. 19K17847) and National Center for Global Health and Medicine. KT was supported in part by KAKENHI Grants from MEXT/JSPS (grant nos. 18H02845 and 18K19570), National Center for Global Health and Medicine (grant nos. 26-001 and 19A2002), AMED (grant nos. JP18ck0106444, JP18ae0201014, JP20bm0704042, and JP20gm1210011), Ono Medical Research Foundation, Kanzawa Medical Research Foundation, and Takeda Science Foundation.&#160;

The protocol follows the guidelines of National Center for Global Health and Medicine. All experimental procedures performed on mice are approved by the National Center for Global Health and Medicine animal experiment committee. 
NOTE: An overview of the protocol is illustrated in Figure 1.
1. Lipid preparation
<ol>
	<li>Dissolve the following lipids in methanol in glass tubes at the indicated concentrations: sodium palmitate, 16 mg/mL; sodium oleate, 30 mg/mL; and cholesterol, 4 mg/mL. Store the lipid solution at -30 &#176;C and thaw the sample before use.</li>
	<li>Mix the lipid solutions prepared in step 1.1 in a fresh glass tube at the doses necessary to obtain the final concentration of 100 &#181;g/mL palmitate, 100 &#181;g/mL oleate, and 20 &#181;g/mL cholesterol. For instance, when preparing 10 mL of culture media, mix 62.5 &#181;L of palmitate solution, 33 &#181;L of oleate solution, and 50 &#181;L of cholesterol solution in the glass tube.</li>
	<li>Evaporate the methanol by passing nitrogen gas through the lipid solution (Figure 2A and 2B). If nitrogen gas is not available, pass air through the solution by using a pipette-aid.</li>
	<li>Completely evaporate the remaining methanol by heating the glass tube in a water bath at 37 &#176;C (Figure 2C). 
	&#8203;NOTE: The use of a high concentration of N2 gas in confined space is potentially harmful. Although the volume used in the protocol to evaporate methanol is limited and thus is considered as safe, ventilating the room adequately and labeling areas of potentially high concentrations of N2 gas is important should an unexpected gas leak occur.</li>
</ol>
2. Medium preparation
<ol>
	<li>Prepare Dulbecco&#39;s modified Eagle medium (DMEM)/F12 medium (with HEPES and glutamine). Add penicillin and streptomycin sulfate to the medium to the final concentration of 50 units and 50 &#181;g/mL, respectively. DMEM/F12 medium containing antibiotics can be stored at 4 &#176;C for at least 2 months.</li>
	<li>Add 4% w/v of bovine serum albumin (BSA) to Dulbecco&#39;s modified Eagle medium (DMEM)/F12 medium (with HEPES and glutamine).</li>
	<li>Adjust the pH of the medium to pH 7.4-7.8 using NaOH solution, typically to pH 7.6.</li>
	<li>Add the medium to the glass tube prepared in step 1. To achieve a solution with maximum solubility, addition of 3-15 mL medium is recommended.</li>
	<li>Completely dissolve the lipids by sonication (optimal: more than 20 min of sonication). After the BSA and lipids are dissolved, the sample should be stored at -80 &#176;C and used within 2 months. Thaw immediately before use.</li>
	<li>Add 1/1,000 of the insulin, transferrin, sodium selenite, and ethanol amine (ITS-X) mixture to the DMEM/F12. Filter the mixed medium using a 0.22 &#181;m filter (designated as &#34;culture media&#34;). Before use, add human stem cell factor (SCF) and human thrombopoietin (TPO) to the culture media at a final concentration of 3 ng/mL each. After adding the cytokines and ITS-X, the medium cannot be stored.</li>
	<li>Staining buffer: Add 10% FCS to Ca- and Mg-free phosphate-buffered saline (PBS). This solution can be stored at 4 &#176;C for 2 weeks.</li>
	<li>Thawing media: Add 10% FCS to the DMEM/F12. This solution is single-use.</li>
	<li>Cytokine stock solution: Dissolve each human SCF or human TPO in PBS (Ca- and Mg-free) at 20 &#181;g/mL. This solution can be stored at -80 &#176;C for at least one year without a loss of activity. Once thawed, store the stock solution at 4 &#176;C and use within 1 month. 
	&#8203;NOTE: Examine the lot after every purchase to avoid variation in the contaminants in BSA. Culture HSCs at the same time with different batches of BSA and examine the phenotypic HSC number at day 7 as described below. If a BSA batch shows a low number or frequency of CD34+ cells (e.g. less than a half the number of input cells), avoid using this batch.</li>
</ol>
3. Preparation of human bone marrow CD34+ cells
<ol>
	<li>Purchase human CD34+ bone marrow cells and store the cells in liquid nitrogen until use. Alternatively, bone marrow cells from a healthy volunteer or fresh cord blood cells can be used upon approval by the institute ethical committee and donor consent.</li>
	<li>Warm 10 mL of thawing media in a 15 mL conical tube in a 37 &#176;C water bath.</li>
	<li>Thaw the frozen cells in vials in a 37 &#176;C water bath within 2 min. After wiping the vial with 70% ethanol to remove contaminants, transfer the cells to the 15 mL conical tube containing the pre-warmed medium from step 3.2.</li>
	<li>Centrifuge the 15 mL tube at 200 &#215; g for 15 min at room temperature. Aspirate the supernatant carefully to keep the pellet intact (leaving &#60; 50 &#181;L of medium). Resuspend the cells with the remaining medium and transfer them to a 1.5 mL tube on ice. 
	NOTE: Exposure to human-derived samples directly in the mucosa including the eye or wounded tissue may cause infection by known or unknown pathogens; thus, gloves and eyeglasses are recommended when handling human cells. Even if the purchased CD34+ cells test negative for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus 1 (HIV1), careful monitoring should be performed according to institutional guidelines if an exposure incidence occurs. Follow the institutional guidelines when disposing of materials exposed to human cells.</li>
</ol>
4. Sorting HSCs
<ol>
	<li>Label the cells with fluorochrome-conjugated antibodies. Mix 50 &#181;L of staining buffer plus 10 &#181;L of anti-CD34-FITC, 2 &#181;L of anti-CD38-PerCP-Cy5.5, 5 &#181;L of anti-CD90-PE-Cy7, and 10 &#181;L of anti-CD45RA-PE. Resuspend the cell pellet in the antibody mixture. Incubate the cells for 30 min at 4 &#176;C in the dark.</li>
	<li>Add 1 mL of staining buffer to wash the antibodies. Centrifuge the tubes at 340 &#215; g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cell pellet in 0.5 mL of staining buffer + 0.1% propidium iodide. Transfer the suspension into a 5 mL tube using a 40 &#181;m filter.</li>
	<li>Gate the phenotypic HSCs within the PI-CD34+CD38-CD90+CD45RA- fraction using the FACS Aria-IIIu and sort the cells into a 1.5 mL tube filled with 500 &#181;L of culture media (Figure 3). Using the gating strategy shown in Figure 3, ~10% of CD34+ cells are expected to be phenotypic HSCs. Record the sorted cell number to calculate the volume of media to resuspend the cells in step 5.3. 
	NOTE: The gating strategy is performed as described previously16 while omitting the lineage marker staining given the low expression of lineage markers in the CD34+CD38- fraction from healthy individuals17 .</li>
	<li>Centrifuge the sorted cells at 340 &#215; g for 5 min at 4 &#176;C and discard the supernatant. Carefully aspirate the supernatant to ensure that the pellet remains intact.</li>
	<li>Store the sorted cells on ice until culture. 
	&#8203;NOTE: Fluorescence compensation of spectral overlap should be performed during the first experiment.</li>
</ol>
5. Cell culture
<ol>
	<li>Transfer 200 &#181;L of the culture media containing cytokines prepared in step 2 into flat-bottom 96-well plates.</li>
	<li>To avoid evaporation of the medium, fill all unused wells with 100-200 &#181;L of PBS.</li>
	<li>Resuspend the sorted HSCs in culture media without cytokines at 60 cells/&#181;L.</li>
	<li>Aliquot 600 HSCs/well (10 &#181;L of cell suspension) into each well. The cell number can be altered. Fewer than 300 cells will lead to larger technical variation, and thus more wells per condition are needed to detect biological differences. Culturing more than 1000 cells in a single well should be avoided because of cytokine/nutrient deprivation or accumulation of unfavorable cytokines/chemokines.</li>
	<li>Culture the cells in a humidified multi-gas incubator at 37 &#176;C in a 5% CO2 and 1% O2 atmosphere.</li>
	<li>For cultures over 7 days, replacing half the media volume every 3-4 days is recommended. Carefully aspirate 100 &#181;L of media by pipetting and add 100 &#181;L of newly prepared culture media containing cytokines to each well. The culture media should be pre-warmed at 37 &#176;C.</li>
</ol>
6. Analysis of marker phenotypes using flow cytometry
NOTE: Although the cells should be analyzed after 7 days of culture, the culture duration can be changed.
<ol>
	<li>Discard 170 &#181;L of the medium by using an 8-channel pipette.</li>
	<li>Label the cells with the antibody mixture. Mix 0.5 &#181;L of anti-CD34-FITC, 0.1 &#181;L of anti-CD38-PerCP-Cy5.5, 0.25 &#181;L of anti-CD90-PE-Cy7, 0.5 &#181;L of anti-CD45RA-PE, and 9 &#181;L of staining buffer per well. Add 10 &#181;L of the mixture to the 96-well plate and incubate the cells for 30 min at 4 &#176;C in the dark.</li>
	<li>To wash the antibodies, add 100 &#181;L of staining buffer to the wells and centrifuge the plates at 400 &#215; g for 5 min at 4 &#176;C using low acceleration and medium deceleration.</li>
	<li>Carefully aspirate 100 &#181;L of the supernatant to maintain the cells on the bottom of the wells, then resuspend the cells in 200 &#181;L of staining buffer + 0.1% v/v of PI + 1% v/v of fluorescent microspheres (e.g., Flow-Check Fluorospheres).</li>
	<li>Set up the flow cytometry instrument. Acquire data for the samples in fast mode with mixed sample mode on and uptake volumes of 100 &#181;L.</li>
	<li>Export the data in FCS format and analyze it using software such as FlowJo. Cell numbers can be determined using the fluorescent microsphere bead count. For instance, if the researcher adds 2000 beads per well and the bead count is 700, multiply the cell number of each fraction by 2000/700 to estimate the total cell number of the fraction in the well. 
	&#8203;NOTE: Fluorescent microspheres are toxic to cultured cells because of the presence of formaldehyde. This may induce cell death during analysis. Minimize the number of wells (&#60;50 wells) analyzed continuously to avoid bias.</li>
</ol>
7. Transplantation of human HSCs
NOTE: Repopulation activity of cultured cells are validated by transplantation to immunodeficient mice. All procedures must be approved by animal experiment committees or their equivalents.
<ol>
	<li>As donors, prepare a sufficient number of 8-12-week immunodeficient NOD-SCID-Il2rg-null (NOG) mice. As NOG mice are highly susceptible to infection, keep the breeding cages as clean as possible and feed the mice with a sterilized diet and water.</li>
	<li>Culture an adequate number of HSCs for transplantation as described in step 5. For instance, when 5000 HSCs (day 0 equivalent) are transplanted to 6 recipient mice, culture 35,000-40,000 HSCs in 40 wells of a 96-well plate (200 &#181;L of culture media per well) or in 8 wells of a 24-well plate (1 mL of culture media per well). Culture the cells for 2 weeks with half-media changes every 3-4 days in a humidified multi-gas incubator at 37 &#176;C with 5% CO2 and 1% O2. The culture duration can be altered.</li>
	<li>Irradiate NOG mice at 2.5 Gy at 6-24 h prior to transplantation.</li>
	<li>Collect the cultured HSCs in 1.5 mL tubes. Centrifuge the tubes at 340 &#215;g for 5 min at 4&#176;C.</li>
	<li>Carefully aspirate the supernatants and resuspend the cells in sterile ice-cold staining buffer at a cell density of 5000 HSCs (day 0 equivalent) per 200 &#181;L. Transfer this suspension to 3 mL polypropylene tubes. To sterilize the staining buffer, filter it using a 0.22 &#181;m filter.</li>
	<li>Before transplantation, anesthetize the mice by sevoflurane or isoflurane inhalation.</li>
	<li>To transplant 5000 of cultured HSCs (day 0 equivalent), inject 200 &#181;L of the cell suspension into the tail vein or retro orbital sinus of NOG mice irradiated in step 7.1 using a 1 mL syringe and 27-gauge needle. Freshly isolated or thawed bone marrow HSCs can be transplanted as a control. Gloves should be sterilized with 70% v/v ethanol between each procedure.</li>
</ol>
8. Analysis of the frequency of human-derived cells in the peripheral blood
<ol>
	<li>To examine the repopulation of human cells, collect the peripheral blood at 1, 2, and 3 months after transplantation.</li>
	<li>Before collecting the peripheral blood, anesthetize the mice by sevoflurane inhalation.</li>
	<li>Collect 40-80 &#181;L of peripheral blood from the retro-orbital sinus using heparinized glass capillary tubes and suspend this sample in 1 mL of PBS + heparin (1 U/mL) in 1.5 mL tubes.</li>
	<li>Centrifuge the blood suspension at 340 &#215; g for 3 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 1 mL of PBS + 1.2% w/v dextran (200 kDa) for 45 min at room temperature.</li>
	<li>Transfer the supernatant to another 1.5 mL tube and centrifuge at 340 &#215; g for 3 min.</li>
	<li>For red blood cell lysis, resuspend the cells in 0.17 M NH4Cl for 5 min.</li>
	<li>Centrifuge the cell suspension 340 &#215; g for 3 min at 4 &#176;C. Resuspend the cells in 50 &#181;L of staining buffer containing 0.3 &#181;L of anti-mouse Fc-block. Incubate this sample at 4 &#176;C for 5 min.</li>
	<li>Add the following antibodies for surface marker staining: 0.3 &#181;L of mouse CD45-PE-Cy7, 0.3 &#181;L mouse Ter-119-PE-Cy7, 0.3 &#181;L of human CD45-BV421, 0.3 &#181;L of human CD13-PE, 1.2 &#181;L of human CD33-PE, 0.3 &#181;L of human CD19-APC, 0.3 &#181;L of human CD3-APC-Cy7. Incubate the cells at 4 &#176;C for 15 min.</li>
	<li>Wash once with 1 mL of staining buffer and centrifuge at 340 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the cells in 200 &#181;L of staining buffer + 0.1% v/v of PI.</li>
	<li>Transfer the cell suspension to a 96-well flat-bottom plate and acquire data using the flow cytometer in fast mode with the mix sample mode on and an uptake volume of 100 &#181;L.</li>
	<li>Export the data in FCS format for analysis using software such as FlowJo.</li>
	<li>Set up the flow cytometer. Acquire data for the samples in fast mode with the mix sample mode on and an uptake volume of 100 &#181;L.</li>
	<li>Export the data in FCS format for analysis using software such as FlowJo.</li>
</ol>

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The authors declare no conflict of interest associated with this study.

Recently, several methods for expanding HSCs with minimal differentiation have been reported18,19,20,21. Although these methods are excellent, HSCs are forced to activate their cell cycles in the presence of high levels of cytokines, which differs from the in vivo situation in which HSCs show minimal cycling. This protocol is useful for maintaining HSCs as quiescent, as observed in vivo, by recapitulating the bone marrow microenvironment.
By culturing human HSCs under low cytokine, lipid-rich, and hypoxic conditions, HSCs showed minimal cycling while maintaining their marker phenotypes. The critical step in this protocol is the preparation of medium containing a high concentration of fatty acids and cholesterol and low cytokine concentrations and culture under hypoxia (Step 1, Step 2, and Step 4). Without cholesterol and/or fatty acids, the maintenance rate of HSCs under low cytokine concentrations decreases22. Culturing of cells under hypoxic conditions is also important, as reported previously23.
The culture conditions were similar to those used for murine HSCs, except for the cytokine concentrations. Murine HSCs survive without TPO, whereas human HSCs require at least 2 ng/mL of TPO with 3 ng/mL of SCF22. As the concentration of TPO is much higher than that in human serum (~100 pg/mL), the conditions used in this protocol may be missing specific factors to support the survival of human HSCs. FLT3 is expressed on human HSCs24. Addition of its ligand FLT3LG slightly decreases the requirement for TPO to maintain HSCs22.
Human HSCs require higher concentrations of cholesterol as compared to murine HSCs, presumably because of the inability to induce the expression of cholesterol-synthesizing enzymes and susceptibility to lipotoxicity under high concentrations (&#62;400 &#181;g/mL) of fatty acids22. Although only the combination of palmitic, oleic, linoleic, and stearic acids was tested, which are abundantly found in the human serum, other combinations of lipids should be evaluated to reduce lipotoxicity and improve the rate of maintaining functional HSCs.
Although the repopulation activity of cultured human HSCs in immunodeficient mice after two weeks of culture has been confirmed22, this culture system does not fully recapitulate the niche functions of HSCs in vivo. The expression of CD45RA has been reported to increase and the repopulation capacity is inferior to that of freshly sorted HSCs22. However, the concentrations of nutrients, such as glucose, amino acid, pyruvate, and insulin, which are added to the medium at supraphysiological levels, can be optimized. Contaminants in BSA may also compromise the maintenance of HSCs18,25. In addition, some cultured cells undergo cell death, whereas others undergo cell division; thus, the maintenance of the total cell number may not indicate the quiescent status of each cell.
Despite these limitations, the culture conditions described in the protocol developed in this study will help advance the research and engineering of HSCs, particularly under near-physiological conditions. Culture conditions that maintain HSCs with minimal differentiation and cycling activity would be suitable for testing biological and chemical compounds specifically acting on HSCs, manipulating HSCs via lentivirus transduction or genome editing without the loss of stemness, and elucidating the initial step of transformation induced by leukemia-associated genes.

Hematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs) coordinately form a reservoir for continuous replenishment of differentiated cells to maintain hematopoiesis throughout life in humans1. Cell cycle quiescence is a prominent feature of HSCs, differentiating them from MPPs2. Conventionally, HSCs are thought to reside at the apex of the hierarchy of the hematopoietic system, producing all differentiated blood cells. This hierarchical model was mostly deduced from transplantation experiments3. However, recent studies indicated that the dynamics of HSCs differ in vivo as compared to those in transplantation experiments4,5,6,7. Lineage tracing experiments using several barcoding systems revealed that phenotypic murine HSCs are not a unique cell type that contributes to steady-state hematopoiesis, and MPPs, which display limited self-renewal activity upon transplantation settings, continuously supply mature blood cells4,5,8. In contrast, the contribution of HSCs to mature cells is enhanced after bone marrow injury4. This may be attributed to drastic alterations in the microenvironment following bone marrow ablation, including bone marrow transplantation. Although applying lineage tracing of murine cells to human cells is difficult, phylogenetic analysis combining single-cell-derived colony isolation and whole genome sequencing revealed a similar property of the hematopoietic system, in which both HSCs and MPPs are responsible for the daily production of mature cells7. Thus, although transplantation is essential for examining murine or human HSC activity, other experimental models are needed to understand the behaviors of HSCs under physiological conditions.
Culturing methods for HSCs have been studied in detail to understand their clinical applications and characteristics. Human HSCs can be expanded in vitro using a combination of cytokines, reconstitution of extracellular matrices, removal of self-renewal antagonists, co-culture with mesenchymal or endothelial cells, addition of albumin or its replacement, transduction of self-renewal transcription factors, and addition of small-molecule compounds9,10. Some of these methods, including addition of small compounds SR111 and UM17112, have been tested in clinical trials with promising results9. Considering the quiescent nature of HSCs&#160;in vivo, maintaining HSCs with minimal cell cycling is critical for recapitulating HSC behavior in vitro. Quiescent and proliferating HSCs exhibit differential cell cycle entry13, metabolic status14, and tolerance against multiple stresses15 . The methods used to maintain the quiescence of human HSCs in vitro are limited.
By mimicking the microenvironment of the bone marrow (hypoxic and rich in lipids) and optimizing the concentration of cytokines, human HSCs can be maintained undifferentiated and quiescent under culture. Recapitulating the quiescent nature of HSCs in vitro will improve the understanding of the steady state properties of HSCs and enable experimental manipulation of HSCs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Human bone marrow CD34+ progenitor cells</td>
			<td>Lonza</td>
			<td>2M-101C</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD/Shi-scid,IL-2R&#947;KO Jic</td>
			<td>In-Vivo Science Inc.</td>
			<td>https://www.invivoscience.com/en/nog_mouse.html</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD34-FITC (clone: 581)</td>
			<td>BD biosciences</td>
			<td>Cat# 560942; RRID: AB_10562559</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD38-PerCP-Cy5.5&#160;</td>
			<td>BD biosciences</td>
			<td>Cat# 551400; RRID: AB_394184</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD45RA-PE</td>
			<td>BD biosciences</td>
			<td>Cat# 555489; RRID: AB_395880</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD90-PE-Cy7 (clone: 5E10)</td>
			<td>BD biosciences</td>
			<td>Cat# 561558; RRID: AB_10714644</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD13-PE (clone: WM15)</td>
			<td>BioLegend</td>
			<td>Cat# 301703; RRID: AB_314179</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD33-PE (clone: WM53)</td>
			<td>BD biosciences</td>
			<td>Cat# 561816; RRID: AB_10896480</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD19-APC (clone: SJ259)</td>
			<td>BioLegend</td>
			<td>Cat# 363005; RRID: AB_2564127</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD3-APC-Cy7 (clone: SK7)</td>
			<td>BD biosciences</td>
			<td>Cat# 561800; RRID: AB_10895381</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-human CD45-BV421 (clone: HI30)</td>
			<td>BD biosciences</td>
			<td>Cat# 563880; RRID:AB_2744402</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse CD45-PE-Cy7 (clone: 30-F11)</td>
			<td>BioLegend</td>
			<td>Cat# 103114; RRID: AB_312979</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse Ter-119-PE-Cy7 (clone: TER-119)</td>
			<td>BD biosciences</td>
			<td>Cat# 557853; RRID: AB_396898</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (anti-mouse CD16/32) (clone: 2.4-G2)</td>
			<td>BD Biosciences</td>
			<td>Cat# 553142; RRID: AB_394657</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Nacalai Tesque</td>
			<td>Cat# 14249-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS-X</td>
			<td>Thermo Fisher Scientific</td>
			<td>Cat# 51500056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Meiji Seika</td>
			<td>PGLD755</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin sulfate</td>
			<td>Meiji Seika</td>
			<td>SSDN1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma Aldrich</td>
			<td>Cat# A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium palmitate</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>Cat# P0007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium oleate</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>Cat# O0057</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>Cat# C0318</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride</td>
			<td>Fujifilm</td>
			<td>Cat# 017-2995</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydrogen Carbonate</td>
			<td>Fujifilm</td>
			<td>Cat# 191-01305</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA&#65381;2Na</td>
			<td>Fujifilm</td>
			<td>Cat# 345-01865</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparine Na</td>
			<td>MOCHIDA PHARMACEUTICAL CO.,&#160;LTD.</td>
			<td>Cat# 224122557</td>
			<td></td>
		</tr>
		<tr>
			<td>Sevoflurane</td>
			<td>Fujifilm</td>
			<td>Cat# 193-17791</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextran</td>
			<td>Nacalai Tesque</td>
			<td>Cat#&#160; 10927-54</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human SCF</td>
			<td>PeproTech</td>
			<td>Cat# 300-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human TPO</td>
			<td>PeproTech</td>
			<td>Cat# 300-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Flt3 ligand</td>
			<td>PeproTech</td>
			<td>Cat# 300-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Life Technologies</td>
			<td>Cat# P3566</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow-Check Fluorospheres</td>
			<td>Beckman Coulter</td>
			<td>Cat# 7547053</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo version 10</td>
			<td>BD Biosciences</td>
			<td>https://www.flowjo.com/solutions/flowjo</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro</td>
			<td>Miltenyi Biotec</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Aria3u</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>VELVO-CLEAR VS-25 (sonicator)</td>
			<td>VELVO-CLEAR</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen gas cylinder</td>
			<td>KOIKE SANSHO CO., LTD</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas regulator</td>
			<td>Astec</td>
			<td>Cat# IM-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Multigas incubator</td>
			<td>Astec</td>
			<td>Cat# SMA-30DR</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tube, 16 mL</td>
			<td>Maruemu corporation</td>
			<td>N-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass tube, 50 mL</td>
			<td>Maruemu corporation</td>
			<td>NX-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP Syringe Filter Unit, 0.22&#160;&#181;m, polyethersulfone, 33&#160;mm, gamma sterilized</td>
			<td>Merck Millipore</td>
			<td>SLGPR33RS</td>
			<td></td>
		</tr>
	</tbody>
</table>

a culture method to maintain quiescent human hematopoietic stem cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

After 7 days of culturing the purified HSCs, up to 80% of cells displayed marker phenotypes of CD34+CD38- (Figure 4A). The total cell number depended on the cytokine concentration (Figure 4B). Higher concentrations of SCF and TPO induced entry into the cell cycle, proliferation, and differentiation (Figure 4B). The number of phenotypic HSCs characterized by the marker phenotypes of CD34+CD38-CD90+CD45RA- increased in proportion to the SCF or TPO concentrations (Figure 4B), whereas the frequency among the total cells decreased (Figure 4C). Total cell numbers were equal in the 1.5 ng/mL SCF and 4 ng/mL TPO and the 3 ng/mL SCF 3 and 2 ng/mL TPO combinations, suggesting that HSCs are quiescent during minimal cell cycle activation. Given the individual differences, cytokine titration is recommended for each bone marrow donor.
Following 3 months of transplantation of cultured adult bone marrow HSCs, reconstitution can be evaluated as their frequency in the peripheral blood of human CD45+ murine CD45- Ter119- cells. Three lineages including CD19+ B cells, CD13/CD33+ myeloid cells, and CD3+ T cells were reconstituted in NOG mice transplanted with either freshly thawed HSCs or cultured HSCs (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61938/61938fig01.jpg" /> 
Figure 1. Overview of the procedure. Graphical summary of the procedure involved sorting, culturing, and analyzing human hematopoietic stem cells (HSCs). <a href="https://www.jove.com/files/ftp_upload/61938/61938fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61938/61938fig02.jpg" /> 
Figure 2.Procedure to evaporate methanol from the lipid solution. A) Nitrogen gas cylinder with a gas regulator. B) Procedure for passing nitrogen gas through the lipid solution dissolved in methanol. C) Lipids adhering to the bottom of the glass tube. <a href="https://www.jove.com/files/ftp_upload/61938/61938fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61938/61938fig03.jpg" /> 
Figure 3. Gating strategy for sorting HSCs. The plots show gated CD34+CD38-CD90+CD45RA- cells. Approximately 10% of CD34+ cells were phenotypic HSCs. <a href="https://www.jove.com/files/ftp_upload/61938/61938fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61938/61938fig04.jpg" /> 
Figure 4. Representative cell numbers after 7 days of culturing. A) Representative fluorescence-activated cell sorting plot after culturing the HSCs in SCF (3 ng/mL) and TPO (2 ng/mL). Fraction 1 was enriched for live cells, fraction 2 was enriched for dead cells, and fraction 3 was enriched for debris. Numbers colored in pink indicate the frequency (%) of the gated fraction. B) Number of all HSCs, CD34+CD38- cells, and CD34+CD38-CD90+CD45RA- cells after culturing 600 HSCs under the indicated cytokine conditions. Red dashed lines indicate the initial number of input cells. S: SCF, T: TPO. Mean &#177; standard deviation, n = 4. Numbers following S and T indicate the concentration (ng/mL) of each cytokine. C) Frequency of CD34+CD38- and CD34+CD38-CD90+CD45RA- cells under the indicated cytokine conditions. S: SCF, T: TPO. Mean &#177; standard deviation, n = 4. The error bars for cells cultured in SCF (1 ng/mL) and TPO (0 ng/mL) were omitted because of their high values (37.7 and 47.9, respectively). <a href="https://www.jove.com/files/ftp_upload/61938/61938fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61938/61938fig05.jpg" /> 
Figure 5. Representative FACS plots of donor mice following 3 months of transplantation. A total of 5000 freshly thawed (upper panels) and 2-week cultured HSCs (3 ng/mL SCF and 3 ng/mL TPO; lower panels) were transplanted into NOG mice. hCD19 marks human B cells, hCD13 and hCD33 mark human myeloid cells, and hCD3 marks human T cells.&#160;<a href="https://www.jove.com/files/ftp_upload/61938/61938fig05large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells at JoVE.com

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

a-culture-method-to-maintain-quiescent-human-hematopoietic-stem-cells

Research

JoVE Journal

Bioengineering

4.5K Views.  National Center for Global Health and Medicine. This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Video: A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. These include the limited number of cadaveric pancreas donors and the long-term use of immunosuppressants. Mesenchymal stem cells (MSCs) have been considered to be a potential candidate as an alternative source of islet-like cell generation. Our previous reports have successfully illustrated the establishment of induction protocols for differentiating human dental pulp stem cells (hDPSCs) to insulin-producing cells (IPCs). However, the induction efficiency varied greatly. In this paper, we demonstrate the comparison of hDPSCs pancreatic induction efficiency via integrative (microenvironmental and genetic manipulation) and non-integrative (microenvironmental manipulation) induction protocols for delivering hDPSC-derived IPCs (hDPSC-IPCs). The results suggest distinct induction efficiency for both the induction approaches in terms of 3-dimensional colony structure, yield, pancreatic mRNA markers, and functional property upon multi-dosage glucose challenge. These findings will support the future establishment of a clinically applicable IPCs and pancreatic lineage production platform.

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages

This protocol presents a comparison between two different induction protocols for differentiating human dental pulp stem cells (hDPSCs) toward pancreatic lineages in vitro: the integrative protocol and the non-integrative protocol. The integrative protocol generates more insulin producing cells (IPCs).

As of 2000, the success of pancreatic islet transplantation using the Edmonton protocol to treat type I diabetes mellitus still faced some obstacles. ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

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Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(ε-caprolactone) Electrospun Yarns

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

In vitro Induction of Human Dental Pulp Stem Cells Toward Pancreatic Lineages