Vol 46, No 2 (2008)
Original paper
Submitted: 2011-12-19
Published online: 2008-06-04
An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).
Maciej Halasa, Magdalena Baskiewicz-Masiuk, Elzbieta Dabkowska, Bogusław Machalinski
DOI: 10.2478/v10042-008-0036-1
·
Folia Histochem Cytobiol 2008;46(2):239-243.
Vol 46, No 2 (2008)
ORIGINAL PAPERS
Submitted: 2011-12-19
Published online: 2008-06-04
Abstract
The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei).
Abstract
The identification in murine bone marrow (BM) of very small embryonic-like (VSEL) stem cells, possessing several features of pluripotent stem cells, encouraged us to investigate if similar population of cells could be also isolated from the human umbilical cord blood (UCB). Here our approach to purify VSEL from human UCB is described by employing a two step isolation strategy based on i) hypotonic lysis of erythrocytes followed ii) by multi-parameter FACS sorting. Accordingly, first, erythrocytes are removed from the UCB samples by hypotonic ammonium chloride solution and next, the UCB mononuclear cells (UCB MNC) are stained with monoclonal antibodies against all hematopoietic lineages including the common leukocyte antigen CD45. The cells carrying these markers (lin+CD45+) are eliminated from the sort by electronic gating. At the same time the antibodies against CXCR4, CD34 and CD133 are employed as positive markers to enrich the UCB MNC for VSEL. This combined two step approach enables to purify VSEL stem cells, which are small and express mRNA for pluripotent stem cells (PSC) (Oct-4 and Nanog) and tissue-committed stem cells (TCSC) (Nkx2.5/Csx, VE-cadherin and GFAP) similarly to those isolated from the adult BM (3-5 microm cells with large nuclei).
Title
An efficient two-step method to purify very small embryonic-like (VSEL) stem cells from umbilical cord blood (UCB).
Journal
Folia Histochemica et Cytobiologica
Issue
Vol 46, No 2 (2008)
Article type
Original paper
Pages
239-243
Published online
2008-06-04
Page views
3586
Article views/downloads
2276
DOI
10.2478/v10042-008-0036-1
Bibliographic record
Folia Histochem Cytobiol 2008;46(2):239-243.
Authors
Maciej Halasa
Magdalena Baskiewicz-Masiuk
Elzbieta Dabkowska
Bogusław Machalinski
About this article