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

Forfatterne vil gerne takke Wendy Barrett, Rachel Caballero, og Gabriella Ruiz af Maricopa integrerede sundhedssystemer for de identificerede og donerede navlestrengsblod enheder, Mrinalini Kala for assistance med flow cytometry, og homoseksuelle skurke og Christopher seet for rådgivning om ex vivo myeloid differentiering. Dette arbejde blev støttet af midler til S.S. fra National Institutes of Health (R21CA170786 and R01GM127464) og American Cancer Society (den institutionelle forskningsbevilling 74-001-34-IRG). Indholdet er udelukkende ansvaret for forfatterne og ikke nødvendigvis repræsenterer de officielle synspunkter af de nationale institutter for sundhed.

Humant navlestrengen blod til eksperimenter blev doneret af raske personer efter informeret samtykke til Maricopa integrerede sundhedssystemer (MIHS), Phoenix. De deidentificerede enheder blev opnået gennem en materiale overførselsaftale mellem MIHS og University of Arizona.
1. reagenser og buffere
Bemærk: Forbered alle reagenser og buffere under sterile forhold i et biologisk sikkerhedskabinet.
<ol><li>Der forberedes steril PBS-BSA-EDTA (PBE) ...

<ol>
	<li>Hao, Q. L., Shah, A. J., Thiemann, F. T., Smogorzewska, E. M., Crooks, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+comparison+of+CD34+++CD38-+cells+in+cord+blood+and+bone+marrow.">A functional comparison of CD34 + CD38- cells in cord blood and bone marrow.</a> Blood. 86 (10), 3745-3753 (1995).</li><li>Manz, M. G., Miyamoto, T., Akashi, K., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+isolation+of+human+clonogenic+common+myeloid+progenitors.">Prospective isolation of human clonogenic common myeloid progenitors.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (18), 11872-11877 (2002).</li><li>Kondo, M., Weissman, I. L., Akashi, K. <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+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91 (5), 661-672 (1997).</li><li>Seita, J., Weissman, I. 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. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+population+of+CD34++cells+in+cord+blood.">A unique population of CD34+ cells in cord blood.</a> Stem Cells. 13 (2), 158-166 (1995).</li><li>Hordyjewska, A., Popiolek, L., Horecka, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+hematopoietic+stem+cells+of+umbilical+cord+blood.">Characteristics of hematopoietic stem cells of umbilical cord blood.</a> Cytotechnology. 67 (3), 387-396 (2015).</li><li>Bapat, 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=Myeloid+Disease+Mutations+of+Splicing+Factor+SRSF2+Cause+G2-M+Arrest+and+Skewed+Differentiation+of+Human+Hematopoietic+Stem+and+Progenitor+Cells.">Myeloid Disease Mutations of Splicing Factor SRSF2 Cause G2-M Arrest and Skewed Differentiation of Human Hematopoietic Stem and Progenitor Cells.</a> Stem Cells. 36, 1-13 (2018).</li><li>Yip, B. 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. L., Smogorzewska, E. M., Barsky, L. W., Crooks, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+identification+of+single+CD34+CD38-+cells+with+both+lymphoid+and+myeloid+potential.">In vitro identification of single CD34+CD38- cells with both lymphoid and myeloid potential.</a> Blood. 91 (11), 4145-4151 (1998).</li><li>Moretta, F., 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+generation+of+human+innate+lymphoid+cells+is+influenced+by+the+source+of+hematopoietic+stem+cells+and+by+the+use+of+G-CSF.">The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G-CSF.</a> European Journal of Immunology. 46 (5), 1271-1278 (2016).</li><li>Sanz, E., 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=Ordering+human+CD34+CD10-CD19++pre/pro-B-cell+and+CD19-+common+lymphoid+progenitor+stages+in+two+pro-B-cell+development+pathways.">Ordering human CD34+CD10-CD19+ pre/pro-B-cell and CD19- common lymphoid progenitor stages in two pro-B-cell development pathways.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (13), 5925-5930 (2010).</li><li>Egeland, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+differentiation+of+purified+CD34++cells+after+stimulation+with+recombinant+human+granulocyte-monocyte+colony-stimulating+factor+(CSF),+granulocyte-CSF,+and+interleukin-3.">Myeloid differentiation of purified CD34+ cells after stimulation with recombinant human granulocyte-monocyte colony-stimulating factor (CSF), granulocyte-CSF, and interleukin-3.</a> Blood. 78 (12), 3192-3199 (1991).</li><li>Ogawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+and+proliferation+of+hematopoietic+stem+cells.">Differentiation and proliferation of hematopoietic stem cells.</a> Blood. 81 (11), 2844-2853 (1993).</li><li>Perdomo, J., Yan, F., Leung, H. H. L., Chong, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+Differentiation+and+Platelet+Formation+from+Human+Cord+Blood-derived+CD34++Cells.">Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells.</a> Journal of Visualized Experiments. (130), e56420 (2017).</li><li>Palii, C. G., Pasha, R., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral-mediated+knockdown+during+ex+vivo+erythropoiesis+of+human+hematopoietic+stem+cells.">Lentiviral-mediated knockdown during ex vivo erythropoiesis of human hematopoietic stem cells.</a> Journal of Visualized Experiments. (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>

Den her beskrevne protokol er egnet til ex vivo-differentiering af UCB-afledte CD34 + hspc'er til de fire myeloid- lineager. Indledende inkubation med en cytokinblanding bestående af SCF, TPO, Flt3L og IL3 stimulerer CD34+ cellerne. Efterfølgende opnås differentiering med en cocktail af SCF, IL3, Flt3L, EPO og TPO. I denne blanding er SCF, IL3 og Flt3L vigtige for overlevelse og spredning af CD34+ hscs. EPO og TPO fremme differentiering mod erythrocytter og megakaryocytter, henholdsvis...

Normal differentiering af hæmatopoietiske stamceller (HSCs) er afgørende for vedligeholdelse af fysiologiske niveauer af alle blodlegemer lineages. Under differentieringen, i en koordineret respons på ekstracellulære signaler, herunder vækstfaktorer og cytokiner, giver hscs først Multipotente stamceller (MPP), der har lympho-myeloid potentiel1,2,3 ,4 (figur 1). Mpp'er giver anledning til almindeligt myeloid progenitorer (CMPs) og fælles lymfoide progenitorer (Clp'er), som er begrænset til Lineage. Clps differentiere i de lymfoide nedstamningens består af B, T, og naturlige dræber celler. CMPs genererer myeloid nedstamningens gennem to mere begrænsede stamceller, megakaryocyte erytroide progenitorer (MEP'er) og granulocytmonocytter progenitorer (gmps). MEPerne giver anledning til megakaryocytter og erythrocytter, mens GMPs giver anledning til granulocytter og monocytter. Ud over at opstå gennem CMPs er megakaryocytter blevet rapporteret til også at opstå direkte fra hscs eller tidlige mpps via ikke-kanoniske veje5,6.
Hæmatopoietisk frempind og stamceller (Hspc'er) er karakteriseret ved overfladen markør CD34 og manglen på Lineage specifikke markører (Lin-). Andre overflade markører, der almindeligvis anvendes til at skelne mellem HSCs og myeloid stamcellepopulationer, omfatter CD38, CD45RA og CD1232 (figur 1). HSCs og MPPs er henholdsvis Lin-/Cd34+/CD38- og Lin-/cd34+/CD38+. Myeloid bekræftet stamceller populationer er kendetegnet ved tilstedeværelsen eller fravær af CD45RA og CD123. CMPs er Lin-/cd34+/CD38+/CD45RA-/cd123Lo, gmps er Lin-/cd34+/CD38+/CD45RA+/cd123Lo, og MEP'erne er Lin-/cd34+ /Cd38+/Cd45ra-/cd123-.
Den samlede population af CD34- og stamceller kan fås fra humant navlestrengsblod (UCB), knoglemarv og perifert blod. CD34+- celler udgør 0,02% til 1,46% af totale mononukleære celler (multinationale selskaber) i humant UCB, hvorimod deres andel varierer mellem 0,5% og 5,3% i knoglemarv og er meget lavere ved ~ 0,01% i perifert blod7,8,9 . Den proliferativ kapacitet og differentierings potentialet for UCB-afledte CD34+- celler er signifikant højere end for knoglemarv eller perifert blodlegemer1,10, hvilket giver en klar fordel for at opnå tilstrækkeligt materiale til molekylære analyser i kombination med immunophenotypiske og morfologiske karakterisering af cellerne under differentiering.
Ex vivo differentiering af navlestrengen blod afledt CD34+ hspc'er er en bredt anvendt model til undersøgelse af normal hæmatopoiese og hæmatopoietiske sygdomsmekanismer. Når det dyrkes med de relevante cytokiner, kan UCB CD34+ hspc'erne induceres for at skelne langs myeloid eller lymfoid nedstamningens11,12,13,14,15 , 16. her beskriver vi protokoller for isolation og immunophenotypic karakterisering af CD34+ hspc'erne fra Human UCB, og for deres differentiering til myeloid Lineage celler. Dette kultur system er baseret på cytokininduceret differentiering af Hspc'er i nærværelse af MS-5 stromale celler til at efterligne mikromiljøet i knoglemarven. Dyrkningsforholdene forårsager en indledende udvidelse af CD34+ cellerne efterfulgt af deres differentiering til celler, der udtrykker markører for de fire myeloide Lineage celler, nemlig granulocytter (CD66b), MONOCYTTER (CD14), megakaryocytter (CD41), og erythrocytter (CD235a). Anvendelser af CD34+ celle differentierings protokollen omfatter undersøgelser af molekylære mekanismer, der regulerer hæmatopoiesis, og undersøgelser af virkningen af myeloid sygdom associerede mutationer og små molekyler på selvfornyelse og differentiering af Hspc'er.

<table border="0" cellpadding="0" cellspacing="0" style="width:785px;" width="786">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 differentiering af humane hæmatopoietiske stamceller er en meget anvendt model til at studere hæmatopoiese. Den protokol, der er beskrevet her, er for cytokininduceret differentiering af CD34 + hæmatopoietisk stamcelle-og progenitorceller til de fire myeloid- Lineage celler. CD34+ celler isoleres fra humant navlestrengen blod og co-kultiveret med MS-5 stromale celler i nærværelse af cytokiner. Immunophenotypisk karakterisering af stammen og stamceller, og de differentierede myeloide Lineage celler beskrives. Ved hjælp af denne protokol kan CD34+ celler inkuberet med små molekyler eller transduceres med lentivira til at udtrykke myeloide sygdoms mutationer for at undersøge deres indvirkning på myeloid differentiering.

Anvendelsen af ovenstående protokoller giver 5,6 (± 0,5) x 108 multinationale selskaber og 1 (± 0,3) x 106 CD34+ celler fra en ledning blod enhed på ~ 100 ml. Procentdelen af total CD34+ celler svinger mellem 80-90% (figur 2A, B). Immunophenotypisk analyse af ordningen beskrevet af Manz et al.5 viser, at CD34+ cellerne typisk består af ~ 20% hscs og ~ 72% mpps, der er 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

Her præsenterer vi en protokol for immunophenotypic karakterisering og cytokin induceret differentiering af navlestrengsblod afledt CD34+ hæmatopoietisk stilk og stamceller til de fire myeloid lineages. Ansøgningerne i denne protokol omfatter undersøgelser af effekten af myeloid sygdoms mutationer eller små molekyler på myeloid differentiering af CD34+ celler.

Panmyeloid differentiering af humant navlestrengsblod afledt CD34+ hæmatopoietisk stamcelle-og progenitorceller

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

Denne protokol kan bruges til at studere de molekylære og cellulære ændringer, der opstår under den normale myeloid differentiering af humane CD34 positive hæmatopoietiske stængel og progenitor celler. Den største fordel ved denne protokol er, at det giver mulighed for differentiering af CD34 positive celler langs alle fire myeloid slægter, herunder megakaryocytter, erythrocytter, granulocytter, og monocytter. Demonstration af proceduren vil være Aditi Bapat, en post-doc fra mit laboratorium.For mononuklear celle isolering fra en menneskelig navlestrengsblod prøve, sterilisere den ydre overflade af en pose navlestrengsblod med 70% ethanol, før overføre posen indholdet i en steril 250 milliliter plastflaske i en biologisk sikkerhed kabinet. Blodstyndt med et tilsvarende rumtemperatur Hanks buffererede saltvandsopløsning, og der tilsættes 15 milliliter mononuklear cellefraktioneringsmedium til hver af fem sterile koniske rør på 50 milliliter. Efter skånsom blanding hældes omhyggeligt 30 milliliter af det fortyndede navlestrengsblod på det mononukleare fraktioneringsmedium i hvert rør, hvor de 50 milliliterrør holder i en vinkel for at undgå at blande lagene.Cellerne adskilles ved tæthedsgradientuering, og top 15 til 20 milliliter plasma over det mononukleare cellelag fjernes. Brug en pipette til omhyggeligt at overføre det eksponerede mononukleare cellelag til et nyt 50 milliliterrør, der samler de mononukleare celler fra to til tre rør sammen, og bring det endelige volumen i hvert rør op til 50 milliliter med kold PBE-buffer. Pellet de mononukleare celleprøver ved centrifugering, og aspirere supernatanterne.Bank forsigtigt på hvert rør for at løsne cellepillerne, og pellets fortyndes i fem milliliter iskold PBE-buffer. Cellerne fra tre til fire rør kombineres i et 50 milliliter konisk rør, og alle rørene vaskes med fem milliliter frisk PBE-buffer for at indsamle eventuelle resterende celler. Efter optælling bringes det endelige volumen i røret op til 50 milliliter med frisk iskold PBE buffer, og cellerne opsamres ved centrifugering.For at isolere CD34 positive mononukleare celler skal pelleten igen suspenderes i 50 milliliter frisk PBE-buffer, før cellerne indsamles med en anden centrifugering. Efter at supernatanten er kasseret, skal du trykke forsigtigt for at løsne pellet, og re-suspendere cellerne på en gang 10 til de otte mononukleare celler pr 300 mikroliter iskold PBE buffer koncentration. Bloker enhver uspecifikke binding med 100 mikroliter af Fc receptor blokering reagens, bland forsigtigt, og derefter tilføje 100 mikroliter af CD34 mikroperler fra en magnetisk aktiveret celle-sortering menneskelige CD34 mikroperle kit per en gange 10 til de otte celler med blid blanding, og inkubere cellerne i 30 minutter ved fire grader Celsius.Mens cellerne inkuberes, skal du placere en LS-kolonne og et forsepareringsfilter i et MACS-separatormagefelt. Ligevægt kolonnen med to milliliter iskold PBE buffer og pre-separation filter med en milliliter buffer. Lad kolonnen tømmes i et konisk rør på 15 milliliter, og gennemstrømningen kasseres.Ved inkubationens afslutning bringes det endelige volumen af den mononukleare celleprøve op til 15 milliliter med frisk iskold PBE-buffer, og cellerne ved centrifugering af cellerne bringes sammen. Ophæng pelletet igen i en milliliter iskold PBE-buffer, og læg cellerne på forsepareringsfilteret på kolonnen. Skyl røret med tre milliliter frisk iskold PBE-buffer, og tilsæt bufferen til forsepareringsfilteret.Kolonnen vaskes fire gange med yderligere tre milliliter iskold PBE-buffer pr. vask uden filteret, så kolonnen kan løbe helt af mellem hver vask. Efter den endelige vask flyttes kolonnen ind i et nyt 15 milliliterrør, og der kastes fem milliliter frisk iskold PBE gennem kolonnen ind i røret. At bestemme fraktioner af stamceller og progenitor populationer i frisk isolerede CD34 positive hæmatopoietiske stamceller og progenitor celler, indsamle cellerne ved centrifugering og re-suspendere pellet på en gange 10 til de seks celler pr 50 mikroliter af flow cytometri buffer koncentration.Dernæst overføres cellerne i 50 mikroliter aliquots pr. fem milliliter fluorescensaktivt cellesorteringsrør i henhold til den eksperimentelle flowcytometrifarvningsprotokol. Tilsæt den relevante antistofcocktail og levende/død farvning til cellerne, og volumen justeres til 100 mikroliter i alt. Efter 20 minutter i mørke beskyttet mod lys, vaskes cellerne med en milliliter frisk flow cytometri buffer pr rør, og re-suspendere cellerne i 500 mikroliter af frisk flow cytometri buffer pr rør.Analysér derefter cellerne på et flowcytometer i henhold til standardflowcytometriske protokoller. For at differentiere mikroperlen isolerede CD34 positive stamceller og progenitor celler i myeloid afstamning celler, første pels brøndene af en steril, ubestrøget 48-brønd plade med 200 mikroliter pr brønd af rekombinant human fibronectin opløsning i PBS i to timer ved stuetemperatur. Ved inkubationens afslutning kasseres fibronectinopløsningen fra hver brønd, og brøndene blokeres med 200 mikroliter på 2% kvægserumalbumin.Efter 30 minutter ved stuetemperatur vaskes brøndene to gange med 500 mikroliter steril PBS pr. vask. Den nyisolerede CD34-positive celler suspenderes igen med fem gange 10 af de fem celler pr. milliliter varm stimulationsmedium. Dernæst frø 200 mikroliter af celler i hver brønd af fibronectin fragment belagt plade og placere pladen i cellen kultur inkubator i 48 timer.I slutningen af inkubationen, indsamle cellerne med blid pipettering, og vaske brønde med 500 mikroliter af opvarmede Dulbecco modificerede Eagle medium, samle vasker med celle suspension. Efter optælling, re-suspendere cellerne på en 2,5 gange 10 til de fire celler pr en milliliter myeloid differentiering medium koncentration. Lag 200 mikroliter af CD34 positive celler pr brønd i en MS-5 stromale celle seedet 48 godt væv kultur plade.Derefter placere pladen i cellen kultur inkubator, forsigtigt erstatte 100 mikroliter af supernatant i hver brønd med 100 mikroliter af frisk 2X myeloid differentiering medium hver tre til fire dage. På dag 21 høstes cellerne til analyse af myeloid afstamningsmarkørudtrykket ved flowcytometri som netop påvist. Den typiske procentdel af den samlede CD34-positive celler, der er isoleret fra navlestrengsblod ved mikroperleadseparation, hvilket er påvist, viser mellem 80 og 90 % immunophenotypanalyse, at disse CD34-positive celler typisk består af en ca. 20% afstamningsadmide negative CD34 positive CD38 negative hæmatopoietiske stamcellepopulation og en 72% afstamning negativ CD34 positiv CD38 positiv multi-potent progenitorcellepopulation.Inden for multi-potent stamcellepopulation, om 25% af cellerne udtrykker fælles myeloid progenitor markører, omkring 15% udtrykke granulocyt monocyte stamfader markører, og omkring 56% udtrykke megakaryocyte erythroid progenitor markører. Immunophenotyping viser også, at procentdelen af CD34 positive celler reducerer fra omkring 90% ved isolation til mindre end 25% på dag 21. Analyse af CD34 negative population viser en samtidig stigning i procentdelen af celler, der udtrykker modne myeloid afstamning markører.Endvidere viser Wright-Giemsa-farvede celler forskellige morfologiske egenskaber på dag et og 21 med de udifferentierede kulturer, der udviser store, runde kerner og meget lidt cytoplasma og de differentierede kulturer, der udviser morfologiske egenskaber ved afstamningsceller, herunder som er monocytter, granulocytter og erythroblaster. Husk at hælde det fortyndede navlestrengsblod på mononukleare cellefraktioneringsmediet uden at blande lagene, og værre ikke de POSITIVE MS-5- og CD34-celler, når mediet ændres. Efter denne differentieringsprotokol kan CD34-positive cellekulturer analyseres for morfologiske ændringer, apoptotiske virkninger, ændringer i cellecyklusmønstre og graden af differentiering.

Research

JoVE Journal

Developmental Biology

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

Direkte Induktion af humane neurale stamceller fra perifert blod hæmatopoietiske stamceller

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

Isolering af neurale stam- / progenitorceller fra Periventricular Region 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 ...

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

Isolering og differentiering af adipøst afledte stamceller fra Porcine subkutane fedtvæv

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

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Chondrogene differentiering Induktion af adipøst afledte stamceller ved centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

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

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente stamceller

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

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

Differentierende chondrocytter fra perifert blodafledte humane inducerede pluripotente stamceller

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

Kondrogen pelletsdannelse fra ledningsblod-afledte inducerede pluripotente stamceller

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