The authors would like to thank Wendy Barrett, Rachel Caballero, and Gabriella Ruiz of Maricopa Integrated Health Systems for the de-identified and donated cord blood units, Mrinalini Kala for assistance with flow cytometry, and Gay Crooks and Christopher Seet for advice on ex vivo myeloid differentiation. This work was supported by funds to S.S. from the National Institutes of Health (R21CA170786 and R01GM127464) and the American Cancer Society (the Institutional Research Grant 74-001-34-IRG). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Human umbilical cord blood for experimentation was donated by healthy individuals after informed consent to Maricopa Integrated Health Systems (MIHS), Phoenix. The deidentified units were obtained through a Material Transfer Agreement between MIHS and the University of Arizona.
1. Reagents and Buffers
NOTE: Prepare all reagents and buffers under sterile conditions in a biological safety cabinet.
<ol>
	<li>Prepare sterile PBS-BSA-EDTA (PBE) buffer by adding 7.2 mL of sterile 35% bovine serum albumin (BSA; use sterile tissue culture grade 35% BSA) and 5 mL of sterile 0.5 M ethylene diamine tetra acetic acid (EDTA) to 487.8 mL of sterile Dulbecco&#39;s phosphate buffered saline (DPBS). Filter sterilize and de-gas the buffer by leaving the filter attached to vacuum in a biological safety cabinet for at least 2 h. Aliquot the de-gassed buffer in smaller volumes (250 mL) and store at 4 &#176;C.</li>
	<li>Prepare 1x Hanks balanced salt solution (1x HBSS). Dilute 100 mL of 10x HBSS stock in 900 mL of sterile distilled water to make 1 L of 1x HBSS and adjust pH to 7.2. Filter sterilize the 1x HBSS before use.</li>
	<li>Prepare 2% acetic acid solution by adding 2 mL of glacial acetic acid to 98 mL of distilled water.</li>
	<li>Prepare 20 ng/mL recombinant human fibronectin fragment solution in 1x PBS. Make 10 mL per 48-well plate.</li>
	<li>Prepare 2% BSA. To 100 mL 1x PBS, add 2 grams of BSA fraction V. Filter sterilize before use.</li>
	<li>Make complete Dulbecco&#8217;s Modified Eagle medium (DMEM) by adding the DMEM powder, 3.7 gm sodium bicarbonate, 100 mL fetal bovine serum (FBS) and 5 mL penicillin-streptomycin (PS) to 800 mL of sterile distilled water. Mix and make up the volume to 1 L, and filter sterilize before use.</li>
	<li>Prepare freezing medium by mixing 90 mL of FBS and 10 mL of sterile dimethyl sulfoxide (DMSO).</li>
	<li>Prepare stimulation medium. To serum free medium, add L-glutamine to 2 mM, and cytokines to a final concentration of 50 ng/mL for stem cell factor (SCF), thrombopoietin (TPO), and Fms-like tyrosine kinase 3 ligand (Flt3L), and 20 ng/mL for interleukin-3 (IL3) (see Table of Materials for preparation of cytokine stock solutions). Make 5 mL per 48-well plate.</li>
	<li>Prepare 1x myeloid differentiation medium. To complete DMEM, add cytokines to a final concentration of 5 ng/mL for SCF, Flt3L, and IL3, 50 ng/mL for TPO, and 4 units/mL for erythropoietin (EPO). Make 5 mL per 48-well plate.</li>
	<li>Prepare 2x myeloid differentiation medium. To complete DMEM, add cytokines to a final concentration 10 ng/mL for SCF, Flt3L, and IL3, 100 ng/mL for TPO, and 8&#160;units/mL for EPO. Make 5 mL per 48-well plate.</li>
	<li>Prepare flow cytometry buffer. To 1 L of 1x PBS, add 10 g of BSA fraction V.</li>
	<li>Prepare 1% paraformaldehyde in 1x PBS (see Table of Materials for details).</li>
	<li>Prepare poly-L-lysine solution by adding 500 &#181;L of 10 mg/mL poly-L-lysine to 49.5 mL of 1x PBS.</li>
</ol>
2. Isolation of Mononuclear Cells from Umbilical Cord Blood
NOTE: For the protocol described below, cord blood volumes of 90 to 100 mL were used. The isolation of CD34+ cells must be performed under sterile conditions in a biological safety cabinet.
<ol>
	<li>Spray the bag containing UCB with 70% ethanol to sterilize the outer surface before introducing it into the biological safety cabinet. Transfer the UCB into a sterile 250 mL plastic bottle and dilute it 1:1 by adding an equal volume of room temperature (RT) 1x HBSS.</li>
	<li>Dispense 15 mL of MNC fractionation medium&#160;(MFM; see Table of Materials) per tube into approximately six sterile 50 mL conical tubes. Tilt the tube with MFM, and gently pour 30 mL of diluted UCB onto the MFM layer without disturbing it.</li>
	<li>Centrifuge the tubes at 400 x g for 30 min at RT with slow acceleration and no brake.</li>
	<li>After centrifugation, aspirate between 15-20 mL of the plasma above the MNC layer. Gently collect the MNCs with a pipette and transfer to a new 50 mL conical tube. 
	NOTE: MNCs deposit as a white buffy layer at the interface of plasma and MFM, and the red blood cells (RBCs) sediment at the bottom of the conical tube.</li>
	<li>Pool MNCs collected from 2-3 tubes together. Make up the volume of each tube to 50 mL with cold PBE buffer.</li>
	<li>Centrifuge the tubes at 300 x g for 10 min at 4 &#176;C, with the brake on low.</li>
	<li>Aspirate the supernatant and tap the tube gently to loosen the cell pellet.</li>
	<li>Re-suspend the pelleted cells in 5 mL of ice cold PBE buffer. Use the same 5 mL of re-suspended cells to collect cells from other tubes. Combine cells from 3 to 4 tubes together in one 50 mL conical tube.</li>
	<li>To collect any remaining cells, wash all the tubes with 5 mL of cold PBE buffer and add to the 50 mL conical tube.</li>
	<li>Count the MNCs by diluting 10 &#181;L of cell suspension into 490 &#181;L acetic acid solution. 
	NOTE: Acetic acid lyses any contaminating RBCs to allow easier counting of MNCs.</li>
	<li>Make up the volume of the cell suspension to 50 mL with ice-cold PBE buffer. Centrifuge the cells at 300 x g for 10 min at 4 &#176;C, with brake on low. 
	NOTE: The MNC suspension may be processed immediately for isolation of CD34+ cells or frozen for later applications.</li>
	<li>To freeze the MNCs, remove the supernatant and re-suspend at a density of 2 x 107 cells/mL in freezing medium at RT.</li>
	<li>Freeze the cells at -80 &#176;C overnight in a cell freezing container and then transfer them to liquid nitrogen.</li>
</ol>
3. Isolation of CD34+ Cells from Mononuclear Cells
<ol>
	<li>If using freshly isolated MNCs proceed directly to step 3.3.</li>
	<li>If using frozen MNCs, quickly thaw the frozen cells in a 37 &#176;C water bath and transfer to a 50 mL conical tube. Collect any remaining cells in the vials with 1 mL of ice-cold PBE buffer and transfer to the 50 mL conical tube.</li>
	<li>Make up the volume of the MNC suspension to 50 mL with cold PBE buffer and centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and tap the tube gently to loosen the cell pellet. Re-suspend the MNCs to 1 x 108 cells in 300 &#181;L of ice-cold PBE buffer.</li>
	<li>Add 100 &#181;L of FcR blocking reagent from the magnetic-activated cell sorting (MACS) human CD34 microbead kit per 1 x 108 MNCs and mix gently. 
	NOTE: This blocks nonspecific binding to the CD34 microbeads.</li>
	<li>Add 100 &#181;L of the CD34 microbeads per 1 x 108 cells. Mix gently and incubate at 4 &#176;C for 30 min in a refrigerator. Do not incubate on ice.</li>
	<li>While the cells are incubating, place an LS column and pre-separation filter in the MACS separator magnetic field. Equilibrate the column with ice-cold PBE buffer by adding 2 mL directly into column and 1 mL in the pre-separation filter. Allow the column to empty into a 15 mL conical tube and discard the flow through.</li>
	<li>Remove the MNCs from 4 &#176;C, add ice-cold PBE buffer to make up the volume to 15 mL, and centrifuge the cells at 300 x g for 10 min at 4 &#176;C, with the brake on low.</li>
	<li>Aspirate the supernatant and re-suspend the cells in 1 mL of ice-cold PBE buffer.</li>
	<li>Add the cell suspension to the pre-separation filter placed on the LS column. 
	NOTE: The pre-separation filter removes any large cell aggregates that may block the column.</li>
	<li>Rinse the tube with 3 mL of ice-cold PBE buffer and add it to pre-separation filter placed on the LS column. After this, discard the pre-separation filter.</li>
	<li>Wash the LS column four times by adding 3 mL of ice-cold PBE buffer, allowing the column to drain each time. 
	NOTE: The CD34+ cells will remain bound to the column and the unlabeled cells will flow through the column.</li>
	<li>After the final wash, remove the column from the MACS separator magnetic field and place it in a new 15 mL conical tube, away from the magnet.</li>
	<li>Add 5 mL ice-cold PBE buffer to the column and expel the bound CD34+ cells by firmly pushing in the plunger.</li>
	<li>Optionally, pass CD34+ cells isolated from the first column over another LS column with the pre-separation filter as described above (steps 3.10&#8722;3.13).</li>
	<li>Count the isolated CD34+ cells with trypan blue (10 &#181;L of cells and 10 &#181;L of trypan blue) to determine the&#160;total number of live cells. 
	NOTE: At this stage, the isolated CD34+ cells may be frozen for later use or may be processed directly for analysis of HSPCs by flow cytometry (section 4) or for differentiation to myeloid lineage cells (section 5).</li>
	<li>Centrifuge the cell suspension at 600 x g for 5 min at RT, with brake on low.</li>
	<li>To freeze the CD34+ cells, aspirate the supernatant and re-suspend up to 5 x 106 cells in 1 mL of freezing medium and freeze as described above (step 2.13). Otherwise, proceed to section 4 or 5.</li>
</ol>
4. Determination of Stem and Progenitor Populations by Flow Cytometry
<ol>
	<li>To determine the fractions of stem and progenitor populations in the freshly isolated CD34+ HSPCs, centrifuge them at 600 x g for 5 min and re-suspend 1 x 106 cells in 50 &#181;L of flow cytometry buffer.</li>
	<li>To 1 x 106 CD34+ cells, add antibodies (see Table of Materials for dilutions) to lineage markers (CD3, CD7, CD10, CD11b, CD19 and CD235a &#8212; all FITC labelled), CD34 (APC-Cy7), CD38 (PE), CD45RA (PE-Cy7), CD123 (APC) and 7-aminoactinomycin D (7-AAD; as a live/dead stain) and make up the total volume to 100 &#181;L. Incubate on ice in the dark for 20 min. 
	NOTE: Fewer than 1 x 106 CD34+ cells may be employed for analysis. If staining fewer cells, the total volume and the volume of antibodies added should be reduced accordingly. For flow cytometry analysis, unstained and single stained MNCs should be used as controls for setting positive and negative gates for each fluorophore.</li>
	<li>After incubation, wash the cells with 1 mL of flow cytometry buffer and centrifuge at 600 x g for 5 min.</li>
	<li>Optionally, fix the stained cells in 0.5 mL of 1% paraformaldehyde solution for 10 min in the dark at RT.</li>
	<li>Centrifuge at 600 x g for 5 min and aspirate the supernatant.</li>
	<li>Wash the fixed/stained cells with 1 mL of flow cytometry buffer and centrifuge at 600 x g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant, re-suspend the cells in 500 &#181;L of flow cytometry buffer, and analyze by a flow cytometer (Table of Materials).</li>
</ol>
5. Myeloid Differentiation of the CD34+ Hematopoietic Stem and Progenitor Cells
NOTE: To differentiate the CD34+ HSPCs to the myeloid lineage cells, they are first stimulated in recombinant human fibronectin fragment coated plates and then seeded on a layer of MS-5 stromal cells in myeloid differentiation medium. Differentiation may be monitored each week for 3 weeks based on expression of cell surface markers specific to the four myeloid lineages. During stimulation and differentiation, all incubations are performed at 37 &#176;C and 5% CO2 in a humidified chamber.&#160;All steps should be performed in a biological safety cabinet.
<ol>
	<li>Coat the wells of a sterile uncoated 48-well plate by adding 200 &#181;L/well of recombinant human fibronectin solution in 1x PBS for 2 h at RT.</li>
	<li>After 2 h, carefully remove the recombinant human fibronectin solution and discard.</li>
	<li>Block the wells with 200 &#181;L/well of 2% BSA for 30 min at RT.</li>
	<li>Aspirate the BSA solution and wash the wells twice with 500 &#181;L of sterile 1x PBS. 
	NOTE: Fibronectin fragment coated plates may be stored at 4 &#176;C for up to 2 weeks.</li>
	<li>To stimulate the CD34+ HSPCs, re-suspend the freshly isolated cells (from step 3.17) at a density of 1 x 105 cells/200 &#181;L or 5 x 105 cells/mL of warm 1x stimulation medium.</li>
	<li>If using frozen CD34+ cells, quickly thaw and transfer the cells to a 15 mL conical tube containing warm complete DMEM. Centrifuge at 600 x g for 5 min and re-suspend the cells as described in step 5.5.</li>
	<li>Plate 200 &#181;L of the CD34+ cell suspension per well of the fibronectin fragment coated 48-well plate and incubate for 48 h.</li>
	<li>On the next day, seed 15,000 MS-5 stromal cells in 200 &#181;L of complete DMEM per well of a 48-well tissue culture treated plate and incubate at 37 &#176;C for 24 h.</li>
	<li>After 48 h of stimulation, collect the CD34+ cells by gentle pipetting. Wash the wells with 500 &#181;L of warm DMEM and pool with the cell suspension.</li>
	<li>Count the cells with trypan blue. Centrifuge the suspension at 600 x g for 5 min and re-suspend at a density of 5,000 cells/200 &#181;L of 1x myeloid differentiation medium.</li>
	<li>From the 48-well tissue culture plate seeded with MS-5 stromal cells, gently aspirate the medium without disturbing the cells. Layer 200 &#181;L (5,000 cells) of the CD34+ cell suspension per well of the plate and incubate at 37 &#176;C. 
	NOTE: Culture of MS-5 cells can be maintained in complete DMEM. On the day the CD34+ cells are seeded, the MS-5 cells must be in a uniform layer (80-90% confluence). It is recommended that the MS-5 cells are plated at least 24 hours prior to seeding of the CD34+ cells.&#160;If MS-5 cells are to be plated 48 h or 72 h prior to seeding the CD34+ cells, the cell density should be adjusted accordingly. It is also recommended that the peripheral wells of the 48-well plate be left blank or filled with 200 &#181;L of sterile water to avoid edge effects.</li>
	<li>Perform half-medium change every 3&#8722;4 days by gently removing 100 &#181;L of the medium from the top of each well and replacing it with 100 &#181;L of 2x myeloid differentiation medium per well.</li>
	<li>On day 21, harvest the cells for analysis of myeloid lineage marker expression by flow cytometry. Without disturbing the MS-5 layer, gently pipette the medium and transfer cells to a 5 mL tube. 
	NOTE: For staining and flow cytometry analysis, cells from 1-2 wells are sufficient. Differentiation may also be monitored on days 1, 7, and 14. If performing immunophenotyping on multiple days, the numbers of wells needed for analysis should be calculated accordingly.</li>
	<li>Stain 5 x 105 cells with antibodies to CD34 (APC-Cy7), CD66b (PE-Cy7), CD14 (PE), CD41 (PerCP-Cy5.5), and CD235a (APC) in a total volume of 50 &#181;L. Incubate on ice in the dark for 20 min. Include unstained and single stained MNCs as controls for setting positive and negative gates for each fluorophore, and 7-AAD as a live/dead stain to eliminate dead cells from the analysis.</li>
	<li>Wash and fix (optional) the stained cells as described above (steps 4.3-4.6).</li>
	<li>Re-suspend the cells in 500 &#181;L of flow cytometry buffer and analyze by a flow cytometer.</li>
</ol>
6. Assessment of Cellular Morphology
<ol>
	<li>Clean microscope slides by spraying ethanol and wiping until squeaky.</li>
	<li>Immerse clean slides in poly-L-lysine solution for 5 min at RT and dry for 1 h at RT.</li>
	<li>Resuspend the HSPCs from step 3.14 and differentiated cells from step 5.13 in 10 &#181;L of flow cytometry buffer.</li>
	<li>Transfer the cell suspension to the coated slides and leave them to air-dry. 
	NOTE: Slides from day 1 and day 21 can be stored at RT and stained simultaneously.</li>
	<li>Pour 0.5 mL of the Wright-Giemsa stain on the slide and let stand for 3 min at RT.</li>
	<li>Add equal volume of deionized water and let stand for additional 10 min at RT.</li>
	<li>Wash the slide in deionized water.</li>
	<li>Visualize stained cells at a magnification of 60x or 100x by a microscope.</li>
</ol>

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(53), e2813 (2011).</li><li>Davies, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+ASXL1+impairs+the+granulomonocytic+lineage+potential+of+human+CD34(+)+progenitor+cells.">Silencing of ASXL1 impairs the granulomonocytic lineage potential of human CD34(+) progenitor cells.</a> British Journal of Haematology. 160 (6), 842-850 (2013).</li><li>Caceres, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TP53+suppression+promotes+erythropoiesis+in+del(5q)+MDS,+suggesting+a+targeted+therapeutic+strategy+in+lenalidomide-resistant+patients.">TP53 suppression promotes erythropoiesis in del(5q) MDS, suggesting a targeted therapeutic strategy in lenalidomide-resistant patients.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (40), 16127-16132 (2013).</li><li>Shi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ASXL1+plays+an+important+role+in+erythropoiesis.">ASXL1 plays an important role in erythropoiesis.</a> Scientific Reports. 6, 28789 (2016).</li><li>Mazumdar, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukemia-Associated+Cohesin+Mutants+Dominantly+Enforce+Stem+Cell+Programs+and+Impair+Human+Hematopoietic+Progenitor+Differentiation.">Leukemia-Associated Cohesin Mutants Dominantly Enforce Stem Cell Programs and Impair Human Hematopoietic Progenitor Differentiation.</a> Cell Stem Cell. 17 (6), 675-688 (2015).</li><li>Chung, K. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enforced+expression+of+an+Flt3+internal+tandem+duplication+in+human+CD34++cells+confers+properties+of+self-renewal+and+enhanced+erythropoiesis.">Enforced expression of an Flt3 internal tandem duplication in human CD34+ cells confers properties of self-renewal and enhanced erythropoiesis.</a> Blood. 105 (1), 77-84 (2005).</li><li>Ambrosini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-1beta+inhibits+ILC3+while+favoring+NK-cell+maturation+of+umbilical+cord+blood+CD34(+)+precursors.">IL-1beta inhibits ILC3 while favoring NK-cell maturation of umbilical cord blood CD34(+) precursors.</a> European Journal of Immunology. 45 (7), 2061-2071 (2015).</li><li>Batard, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-(beta)1+maintains+hematopoietic+immaturity+by+a+reversible+negative+control+of+cell+cycle+and+induces+CD34+antigen+up-modulation.">TGF-(beta)1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation.</a> Journal of Cell Science. 113, 383-390 (2000).</li><li>Huang, N., Lou, M., Liu, H., Avila, C., Ma, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+potent+small+molecule+capable+of+regulating+polyploidization,+megakaryocyte+maturation,+and+platelet+production.">Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production.</a> Journal of Hematology &amp; Oncology. 9 (1), 136 (2016).</li></ol>

The authors have nothing to disclose.

The protocol described here is suitable for ex vivo differentiation of UCB derived CD34+ HSPCs to the four myeloid lineages. Initial incubation with a cytokine mix consisting of SCF, TPO, Flt3L and IL3 stimulates the CD34+ cells. Subsequently, differentiation is achieved with a cocktail of SCF, IL3, Flt3L, EPO, and TPO. In this mix, SCF, IL3, and Flt3L are important for survival and proliferation of CD34+ HSCs. EPO and TPO promote differentiation toward erythrocytes and megakaryocytes, re...

Normal differentiation of hematopoietic stem cells (HSCs) is critical for maintenance of physiological levels of all blood cell lineages. During differentiation, in a coordinated response to extracellular cues including growth factors and cytokines, HSCs first give rise to multipotent progenitor (MPP) cells that have lympho-myeloid potential1,2,3,4 (Figure 1). MPPs give rise to common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) that are lineage-restricted. CLPs differentiate into the lymphoid lineages comprised of B, T, and natural killer cells. CMPs generate the myeloid lineages through two more restricted progenitor populations, megakaryocyte erythroid progenitors (MEPs), and granulocyte monocyte progenitors (GMPs). MEPs give rise to megakaryocytes and erythrocytes, whereas GMPs give rise to granulocytes and monocytes. In addition to arising through CMPs, megakaryocytes have been reported to also arise directly from HSCs or early MPPs via non-canonical pathways5,6.
Hematopoietic stem and progenitor cells (HSPCs) are characterized by the surface marker CD34 and the lack of lineage specific markers (Lin-). Other surface markers that are commonly employed to distinguish HSCs and myeloid progenitor populations include CD38, CD45RA, and CD1232 (Figure 1). HSCs and MPPs are Lin-/CD34+/CD38- and Lin-/CD34+/CD38+, respectively. Myeloid committed progenitor populations are distinguished by the presence or absence of CD45RA and CD123. CMPs are Lin-/CD34+/CD38+/CD45RA-/CD123lo, GMPs are Lin-/CD34+/CD38+/CD45RA+/CD123lo, and MEPs are Lin-/CD34+/CD38+/CD45RA-/CD123-.
The total population of CD34+ stem and progenitor cells can be obtained from human umbilical cord blood (UCB), bone marrow, and peripheral blood. CD34+ cells constitute 0.02% to 1.46% of total mononuclear cells (MNCs) in human UCB, whereas their percentage varies between 0.5% and 5.3% in bone marrow and is much lower at ~0.01% in peripheral blood7,8,9. The proliferative capacity and differentiation potential of UCB derived CD34+ cells is significantly higher than that of bone marrow or peripheral blood cells1,10, thereby offering a distinct advantage for obtaining sufficient material for molecular analyses in combination with performing immunophenotypic and morphological characterization of the cells during differentiation.
Ex vivo differentiation of umbilical cord blood derived CD34+ HSPCs is a widely applied model for investigating normal hematopoiesis and hematopoietic disease mechanisms. When cultured with the appropriate cytokines, the UCB CD34+ HSPCs can be induced to differentiate along the myeloid or lymphoid lineages11,12,13,14,15,16. Here, we describe protocols for isolation and immunophenotypic characterization of the CD34+ HSPCs from human UCB, and for their differentiation to myeloid lineage cells. This culture system is based on cytokine-induced differentiation of HSPCs in the presence of MS-5 stromal cells to mimic the microenvironment in bone marrow. The culture conditions cause an initial expansion of the CD34+ cells, followed by their differentiation to cells that express markers for the four myeloid lineage cells, namely granulocytes (CD66b), monocytes (CD14), megakaryocytes (CD41), and erythrocytes (CD235a). Applications of the CD34+ cell&#160;differentiation protocol include studies on molecular mechanisms regulating hematopoiesis, and investigations of the impact of myeloid disease associated mutations and small molecules on self-renewal and differentiation of HSPCs.

<table border="0" cellpadding="0" cellspacing="0" style="width:785px;" width="786">
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	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">0.4% Trypan blue solution</td>
			<td style="width:112px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250-061</td>
			<td style="width:339px;">Dilute working stock to 0.2% in sterile 1x PBS</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">0.5 M UltraPure Ethylene diamine tetra acetic acid, pH 8.0</td>
			<td style="width:112px;">Gibco&#160;</td>
			<td style="width:119px;">15575-038</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">10x Hanks Balanced Salt Solution (HBSS)</td>
			<td style="width:112px;">Invitrogen</td>
			<td style="width:119px;">14185052</td>
			<td style="width:339px;">Dilute to 1x with sterile distilled water &#38; pH to 7.2</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">2.5% Trypsin, no phenol red</td>
			<td style="width:112px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15090046</td>
			<td style="width:339px;">Dilute working stock to 1x with sterile 1x PBS</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">30 &#181;m Pre-separation filters</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-041-407</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">35% sterile Bovine serum albumin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7979</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">7-AAD</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">420404</td>
			<td style="width:339px;">Used as a live/dead stain to eliminate dead cells from FACS analysis</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD10-FITC antibody (Clone HI10a)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">312207</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Anti-human CD11b-FITC (activated) antibody (Clone CBRM1/5)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">301403</td>
			<td style="width:339px;">Use 1:5 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD123-APC antibody (Clone 6H6)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">306012</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD14-PE antibody (Clone M5E2)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">301806</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD19-FITC antibody (Clone 4G7)</td>
			<td style="width:112px;">BD Biosciences</td>
			<td style="width:119px;">347543</td>
			<td style="width:339px;">Use 1:5 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD235a-APC antibody (Clone GA-R2 (HIR2))</td>
			<td style="width:112px;">BD Biosciences</td>
			<td style="width:119px;">551336</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD235a-FITC antibody (Clone HIR2)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">306609</td>
			<td style="width:339px;">Use 1:50 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD34-APC-Cy7 antibody (Clone 581)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">343514</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD38-PE antibody (Clone HIT2)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">303506</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD3-FITC antibody (Clone UCHT1)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">300405</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD41a-PerCP-Cy5.5 antibody (Clone HIP8)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">303720</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD45Ra-PE-Cy7 antibody (Clone HI100)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">304126</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD66b-PE-Cy7 antibody (Clone G10F5)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">305116</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-human CD7-FITC antibody (Clone CD7-6B7)</td>
			<td style="width:112px;">Biolegend</td>
			<td style="width:119px;">343103</td>
			<td style="width:339px;">Use 1:20 dilution</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">BP231-100</td>
			<td style="width:339px;">Filter sterilize before use</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM) powder with L-Glutamine&#160;</td>
			<td style="width:112px;">Gibco</td>
			<td style="width:119px;">12100046</td>
			<td style="width:339px;">Reconstitute 1 packet to make 1 L of DMEM media&#160; with sodium bicarbonate, 10% FBS &#38; 1% penicillin &#38; streptomycin&#160;</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Fetal bovine serum, Australian source, heat inactivated</td>
			<td style="width:112px;">Omega Scientific</td>
			<td style="width:119px;">FB-22 Lot #609716</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human CD34 microbead kit&#160;</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-046-702</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Human Thrombopoietin (TPO), research grade</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-094-011</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 50 ng/mL for both myeloid differentiation &#38; stimulation medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">L-Glutamine</td>
			<td style="width:112px;">Omega Scientific</td>
			<td style="width:119px;">GS-60</td>
			<td style="width:339px;">2 mM concentration in stimulation medium</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">LS Columns</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-042-401</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MACS Multi stand</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-042-303</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MidiMACS magnetic separator</td>
			<td style="width:112px;">Miltenyi biotech</td>
			<td style="width:119px;">130-042-302</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">MNC fractionation media (Ficol-Paque PLUS)</td>
			<td style="width:112px;">GE Healthcare Biosciences</td>
			<td style="width:119px;">17-1440-03</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">MS-5 cells</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td style="width:339px;">Gift from the laboratory of Gay Crooks, UCLA</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;width:216px;">Paraformaldehyde</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td style="width:339px;">Heat 800 mL of 1x PBS in a glass beaker on a stir plate in a chemical hood to ~65 &#176;C. Add 10 g of paraformaldehyde powder. To completely dissolve the paraformaldehyde, raise the pH by adding 1 N NaOH. Cool and filter the solution and make up the volume to 1 L with 1x PBS. Adjust the pH to 7.2.&#160;</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Penicillin &#38; Streptomycin</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458-100ml</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Poly-L lysine</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2636</td>
			<td style="width:339px;">Make a 10 mg/mL stock in 1x PBS</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human erythropoietin-alpha (rHu EPO-&#945;)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC213-15</td>
			<td style="width:339px;">Make a stock of 2000 units/mL in 1x PBS + 0.1% BSA. Use 4 units/mL for myeloid differentiation</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human fibronectin fragment (RetroNectin)</td>
			<td style="width:112px;">Takara&#160;</td>
			<td style="width:119px;">T100B</td>
			<td style="width:339px;">Use 20 &#181;g/mL diluted in sterile 1x PBS to coat wells prior to stimulation of CD34+ HSCs.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human Flt-3 ligand (rHu Flt-3L)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC214-16</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation &#38; 50 ng/mL in stimulation medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human interleukin-3 (rHu IL-3)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC212-14</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation &#38; 20 ng/mL in stimulation medium</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Recombinant human stem cell factor (rHu SCF)</td>
			<td style="width:112px;">BioBasic</td>
			<td style="width:119px;">RC213-12</td>
			<td style="width:339px;">Make a stock of 100 &#181;g/mL in 1x PBS + 0.1% BSA. Use 5 ng/mL for myeloid differentiation &#38; 50 ng/mL in stimulation medium</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Serum free medium (X-Vivo-15)</td>
			<td style="width:112px;">Lonza&#160;</td>
			<td style="width:119px;">04-418Q</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium bicarbonate</td>
			<td style="width:112px;">Fisher Scientific</td>
			<td style="width:119px;">BP328-500</td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Wright-Giemsa stain, modified</td>
			<td style="width:112px;">Sigma-Aldrich</td>
			<td style="width:119px;">WG16-500</td>
			<td style="width:339px;">Use according to manufacturer&#39;s instructions</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Equipment&#160;</td>
			<td style="width:112px;"></td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">BD LSR II flow cytometer</td>
			<td style="width:112px;">BD Biosciences</td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:112px;">Sorvall Legend RT</td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Light microscope</td>
			<td style="width:112px;">Olympus</td>
			<td style="width:119px;"></td>
			<td style="width:339px;"></td>
		</tr>
	</tbody>
</table>

pan-myeloid differentiation of human cord blood derived cd34+ hematopoietic stem and progenitor cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Application of the above protocols yields 5.6 (&#177; 0.5) x 108 MNCs and 1 (&#177; 0.3) x 106 CD34+ cells from a cord blood unit of ~100 mL. The percentage of total CD34+ cells ranges between 80-90% (Figure 2A,B). Immunophenotypic analysis by the scheme described by Manz et al.5 demonstrates that the CD34+ cells typically consist of ~20% HSCs and ~72% MPPs that are Lin-/CD34+</sup...

Watch this Scientific Journal Video about Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells at JoVE.com

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

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JoVE Journal

Developmental Biology

9.1K Views.  University of Arizona. Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Video: Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally isolated and characterized from bone marrow (BM), then acquired from tissues including adipose tissue, synovium, skin, dental pulp, and fetal appendages such as placenta, umbilical cord blood (UCB), and umbilical cord (UC). MSCs are a heterogeneous cell population with the capacity for (1) adherence to plastic in standard culture conditions, (2) surface marker expression of CD73+/CD90+/CD105+/CD45-/CD34-/CD14-/CD19-/HLA-DR- phenotypes, and (3) trilineage differentiation into adipocytes, osteocytes, and chondrocytes, as currently defined by the International Society for Cellular Therapy (ISCT). Although BM is the most widely used source of MSCs, the invasive nature of BM aspiration ethically limits its accessibility. Proliferation and differentiation capacity of MSCs obtained from BM generally decline with the age of the donor. In contrast, fetal MSCs obtained from UC have advantages such as vigorous proliferation and differentiation capacity. There is no ethical concern for UC sampling, as it is typically regarded as medical waste. Human UC starts to develop with continuing growth of the amniotic cavity at 4-8 weeks of gestation and keeps growing until reaching 50-60 cm in length, and it can be isolated during the whole newborn delivery period. To gain insight into the pathophysiology of intractable diseases, we have used UC-derived MSCs (UC-MSCs) from infants delivered at various gestational ages. In this protocol, we describe the isolation and characterization of UC-MSCs from fetuses/infants at 19-40 weeks of gestation.

Human umbilical cord (UC) can be obtained during the perinatal period as a result of preterm, term, and postterm delivery. In this protocol, we describe the isolation and characterization of UC-derived mesenchymal stem cells (UC-MSCs) from fetuses/infants at 19-40 weeks of gestation.