Name: pan-myeloid differentiation of human cord blood derived cd34+ hematopoietic stem and progenitor cells
Uploaded: 2019-08-09T14:45:00
Description: Watch this Scientific Journal Video about Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells at JoVE.com

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 ...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell:+self-renewal+versus+differentiation.">Hematopoietic stem cell: self-renewal versus differentiation.</a> Wiley Interdisciplinary Reviews: Systems Biology and Medicine. 2 (6), 640-653 (2010).</li><li>Haas, S., 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=Inflammation-Induced+Emergency+Megakaryopoiesis+Driven+by+Hematopoietic+Stem+Cell-like+Megakaryocyte+Progenitors.">Inflammation-Induced Emergency Megakaryopoiesis Driven by Hematopoietic Stem Cell-like Megakaryocyte Progenitors.</a> Cell Stem Cell. 17 (4), 422-434 (2015).</li><li>Sanjuan-Pla, A., 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=Platelet-biased+stem+cells+reside+at+the+apex+of+the+haematopoietic+stem-cell+hierarchy.">Platelet-biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy.</a> Nature. 502 (7470), 232-236 (2013).</li><li>Bender, J. 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=Phenotypic+analysis+and+characterization+of+CD34++cells+from+normal+human+bone+marrow,+cord+blood,+peripheral+blood,+and+mobilized+peripheral+blood+from+patients+undergoing+autologous+stem+cell+transplantation.">Phenotypic analysis and characterization of CD34+ cells from normal human bone marrow, cord blood, peripheral blood, and mobilized peripheral blood from patients undergoing autologous stem cell transplantation.</a> Clinical Immunology and Immunopathology. 70 (1), 10-18 (1994).</li><li>Fritsch, 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=The+composition+of+CD34+subpopulations+differs+between+bone+marrow,+blood+and+cord+blood.">The composition of CD34 subpopulations differs between bone marrow, blood and cord blood.</a> Bone Marrow Transplantation. 17 (2), 169-178 (1996).</li><li>Nimgaonkar, M. 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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=The+U2AF1S34F+mutation+induces+lineage-specific+splicing+alterations+in+myelodysplastic+syndromes.">The U2AF1S34F mutation induces lineage-specific splicing alterations in myelodysplastic syndromes.</a> Journal of Clinical Investigation. 127 (6), 2206-2221 (2017).</li><li>Yoo, E. S., 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=Myeloid+differentiation+of+human+cord+blood+CD34++cells+during+ex+vivo+expansion+using+thrombopoietin,+flt3-ligand+and/or+granulocyte-colony+stimulating+factor.">Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3-ligand and/or granulocyte-colony stimulating factor.</a> British Journal of Haematology. 105 (4), 1034-1040 (1999).</li><li>Hao, Q. 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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. 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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.

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			<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 ...

This protocol can be used to study the molecular and cellular changes that occur during the normal myeloid differentiation of human CD34 positive hematopoietic stem and progenitor cells. The main advantage of this protocol is that it allows the differentiation of CD34 positive cells along all four myeloid lineages including megakaryocytes, erythrocytes, granulocytes, and monocytes. Demonstrating the procedure will be Aditi Bapat, a post-doc from my laboratory.For mononuclear cell isolation from a human umbilical cord blood sample, sterilize the outer surface of a bag of umbilical cord blood with 70%ethanol before transferring the bag contents into a sterile 250 milliliter plastic bottle within a biological safety cabinet. Dilute the blood with an equal volume of room temperature Hank's buffered saline solution, and add 15 milliliters of mononuclear cell fractionation medium to each of five sterile 50 milliliter conical tubes. After gentle mixing, carefully pour 30 milliliters of the diluted umbilical cord blood onto the mononuclear fractionation medium in each tube, holding the 50 milliliter tubes at an angle to help avoid mixing the layers.Separate the cells by density gradient centrifugation, and remove the top 15 to 20 milliliters of plasma above the mononuclear cell layer. Use a pipette to carefully transfer the exposed mononuclear cell layer into a new 50 milliliter tube, pooling the mononuclear cells from two to three tubes together, and bring the final volume in each tube up to 50 milliliters with cold PBE buffer. Pellet the mononuclear cell samples by centrifugation, and aspirate the supernatants.Tap each tube gently to loosen the cell pellets, and dilute the pellets in five milliliters of ice cold PBE buffer. Combine the cells from three to four tubes into one 50 milliliter conical tube, and wash all of the tubes with five milliliters of fresh PBE buffer to collect any remaining cells. After counting, bring the final volume in the tube up to 50 milliliters with fresh ice cold PBE buffer, and collect the cells by centrifugation.To isolate the CD34 positive mononuclear cells, re-suspend the pellet in 50 milliliters of fresh PBE buffer before collecting the cells with another centrifugation. After discarding the supernatant, tap gently to loosen the pellet, and re-suspend the cells at a one times 10 to the eight mononuclear cells per 300 microliters of ice cold PBE buffer concentration. Block any nonspecific binding with 100 microliters of Fc receptor blocking reagent, mix gently, and then add 100 microliters of CD34 microbeads from a magnetic activated cell-sorting human CD34 microbead kit per one times 10 to the eight cells with gentle mixing, and incubate the cells for 30 minutes at four degrees Celsius.While the cells are incubating, place an LS column and pre-separation filter in a MACS separator magnetic field. Equilibrate the column with two milliliters of ice cold PBE buffer and the pre-separation filter with one milliliter of buffer. Allow the column to empty into a 15 milliliter conical tube and discard the flow-through.At the end of the incubation, bring the final volume of the mononuclear cell sample up to 15 milliliters with fresh ice cold PBE buffer and sediment the cells by centrifugation. Re-suspend the pellet in one milliliter of ice cold PBE buffer and load the cells onto the pre-separation filter on the column. Rinse the tube with three milliliters of fresh ice cold PBE buffer and add the buffer to the pre-separation filter.Wash the column four times with an additional three milliliters of ice cold PBE buffer per wash without the filter, allowing the column to fully drain between each wash. After the final wash, move the column into a new 15 milliliter tube, and plunge five milliliters of fresh ice cold PBE through the column into the tube. To determine the fractions of stem and progenitor populations in the freshly isolated CD34 positive hematopoietic stem and progenitor cells, collect the cells by centrifugation and re-suspend the pellet at a one times 10 to the six cells per 50 microliters of flow cytometry buffer concentration.Next, transfer the cells in 50 microliter aliquots per five milliliter fluorescence activated cell-sorting tube according to the experimental flow cytometry staining protocol. Add the appropriate antibody cocktail and live/dead staining to the cells, adjusting the volume to 100 total microliters. After 20 minutes in the dark protected from light, wash the cells with one milliliter of fresh flow cytometry buffer per tube, and re-suspend the cells in 500 microliters of fresh flow cytometry buffer per tube.Then analyze the cells on a flow cytometer according to standard flow cytometric protocols. To differentiate the microbead isolated CD34 positive stem and progenitor cells into myeloid lineage cells, first coat the wells of a sterile, uncoated 48-well plate with 200 microliters per well of recombinant human fibronectin solution in PBS for two hours at room temperature. At the end of the incubation, discard the fibronectin solution from each well and block the wells with 200 microliters of 2%bovine serum albumin.After 30 minutes at room temperature, wash the wells two times with 500 microliters of sterile PBS per wash. Re-suspend the freshly isolated CD34 positive cells at a five times 10 the five cells per milliliter of warm stimulation medium concentration. Next, seed 200 microliters of cells into each well of the fibronectin fragment coated plate and place the plate in the cell culture incubator for 48 hours.At the end of the incubation, collect the cells with gentle pipetting, and wash the wells with 500 microliters of warmed Dulbecco's modified Eagle medium, pooling the washes with the cell suspension. After counting, re-suspend the cells at a 2.5 times 10 to the four cells per one milliliter of myeloid differentiation medium concentration. Layer 200 microliters of CD34 positive cells per well in an MS-5 stromal cell seeded 48 well tissue culture plate.Then place the plate in the cell culture incubator, gently replacing 100 microliters of the supernatant in each well with 100 microliters of fresh 2X myeloid differentiation medium every three to four days. On day 21, harvest the cells for analysis of the myeloid lineage marker expression by flow cytometry as just demonstrated. The typical percentage of total CD34 positive cells isolated from cord blood by microbead separation as demonstrated ranges between 80 to 90%Immunophenotypic analysis reveals that these CD34 positive cells typically consist of an approximately 20%lineage negative CD34 positive CD38 negative hematopoietic stem cell population and a 72%lineage negative CD34 positive CD38 positive multi-potent progenitor cell population.Within the multi-potent progenitor cell population, about 25%of the cells express common myeloid progenitor markers, about 15%express granulocyte monocyte progenitor markers, and about 56%express megakaryocyte erythroid progenitor markers. Immunophenotyping also demonstrates that the percentage of CD34 positive cells reduces from about 90%at isolation to less than 25%on day 21. Analysis of the CD34 negative population demonstrates a concomitant rise in the percentage of cells expressing mature myeloid lineage markers.Further, Wright-Giemsa stained cells demonstrate distinct morphological characteristics on days one and 21 with the undifferentiated cultures exhibiting large, round nuclei and very little cytoplasm and the differentiated cultures displaying morphologic characteristics of lineage cells including which are monocytes, granulocytes, and erythroblasts. Remember to pour the diluted cord blood onto the mononuclear cell fractionation medium without mixing the layers, and do not disturb the MS-5 and CD34 positive cells when changing the medium. Following this differentiation protocol, the CD34 positive cell cultures can be analyzed for morphological changes, apoptotic effects, changes in cell cycle patterns, and the degree of differentiation.

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

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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. ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

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

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