Optimization of isolation and further characterization of umbilical-cord-blood-derived very small embryonic/ epiblast-like stem cells (VSELs).
Because of their small size and density, umbilical cord blood (UCB)-derived very small embryonic/epiblast-like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll-Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three-step isolation protocol that allows recovery of approximately 60% of the initial number of Lin(-)/CD45(-)/CD133(+) UCB-VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133(+) including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin(-)/CD45(-)/CD133(+) cells are sorted by fluorescence-activated cell sorting. The whole isolation procedure takes approximately 2-3 h per UCB unit and isolated cells are highly enriched for an Oct-4(+) and SSEA-4(+) population of small Lin(-)/CD45(-)/CD133(+) cells.
Zuba-Surma
Ewa K
EK
Stem Cell Biology Institute, University of Louisville, KY 40202, USA.
Klich
Iza
I
Greco
Nick
N
Laughlin
Mary J
MJ
Ratajczak
Janina
J
Ratajczak
Mariusz Z
MZ
D000071916:AC133 Antigen; D000643:Ammonium Chloride / Q000494:pharmacology; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics; D018952:Antigens, CD34 / Q000096:biosynthesis / Q000235:genetics; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002499:Centrifugation, Density Gradient; D004912:Erythrocytes / Q000187:drug effects; D005312:Fetal Blood / Q000166:cytology*; D005362:Ficoll; D005434:Flow Cytometry / Q000379:methods*; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007038:Hypotonic Solutions / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted / Q000379:methods; D018189:Immunomagnetic Separation / Q000379:methods*; D016130:Immunophenotyping; D007231:Infant, Newborn; D050814:Octamer Transcription Factor-3 / Q000032:analysis; D010455:Peptides / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000097:blood; D019718:Receptors, CXCR4 / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens / Q000032:analysis
D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; C527276:CXCR4 protein, human; D006023:Glycoproteins; D007038:Hypotonic Solutions; D050814:Octamer Transcription Factor-3; C000606023:PROM1 protein, human; D010455:Peptides; D012333:RNA, Messenger; D019718:Receptors, CXCR4; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D000643:Ammonium Chloride; D005362:Ficoll