Ex vivo identification and characterization of a population of CD13(high) CD105(+) CD45(-) mesenchymal stem cells in human bone marrow.
BACKGROUND
Mesenchymal stem cells (MSCs) are multipotent cells capable of self-renewal and multilineage differentiation. Their multipotential capacity and immunomodulatory properties have led to an increasing interest in their biological properties and therapeutic applications. Currently, the definition of MSCs relies on a combination of phenotypic, morphological and functional characteristics which are typically evaluated upon in vitro expansion, a process that may ultimately lead to modulation of the immunophenotypic, functional and/or genetic features of these cells. Therefore, at present there is great interest in providing markers and phenotypes for direct in vivo and ex vivo identification and isolation of MSCs.


METHODS
Multiparameter flow cytometry immunophenotypic studies were performed on 65 bone marrow (BM) samples for characterization of CD13(high) CD105(+) CD45(-) cells. Isolation and expansion of these cells was performed in a subset of samples in parallel to the expansion of MSCs from mononuclear cells following currently established procedures. The protein expression profile of these cells was further assessed on (paired) primary and in vitro expanded BM MSCs, and their adipogenic, chondrogenic and osteogenic differentiation potential was also determined.


RESULTS
Our results show that the CD13(high) CD105(+) CD45(-) immunophenotype defines a minor subset of cells that are systematically present ex vivo in normal/reactive BM (n = 65) and that display immunophenotypic features, plastic adherence ability, and osteogenic, adipogenic and chondrogenic differentiation capacities fully compatible with those of MSCs. In addition, we also show that in vitro expansion of these cells modulates their immunophenotypic characteristics, including changes in the expression of markers currently used for the definition of MSCs, such as CD105, CD146 and HLA-DR.


CONCLUSIONS
BM MSCs can be identified ex vivo in normal/reactive BM, based on a robust CD13(high) CD105(+) and CD45(-) immunophenotypic profile. Furthermore, in vitro expansion of these cells is associated with significant changes in the immunophenotypic profile of MSCs.
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. c.munizmartin@gmail.com.|Spanish Net on Aging and Frailty (RETICEF) Instituto de Salud Carlos III, Madrid, Spain. c.munizmartin@gmail.com.
Carmen
C
Muñiz
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. c.teodosio@erasmusmc.nl.|Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. c.teodosio@erasmusmc.nl.
Cristina
C
Teodosio
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. amayado@usal.es.
Andrea
A
Mayado
The Molecular Pathology group, Institute of Biomedicine of Seville - Hospital Virgen del Rocio, Seville, Spain. ana.teresa.amaral@gmail.com.
Ana Teresa
AT
Amaral
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. smats@usal.es.
Sergio
S
Matarraz
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. pabarcena@usal.es.
Paloma
P
Bárcena
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. sangar@usal.es.
Maria Luz
ML
Sanchez
Centro de Estudios de Mastocitosis de Castilla La Mancha, Hospital Virgen del Valle, Toledo, Spain. ivana@sescam.jccm.es.
Iván
I
Alvarez-Twose
Hematology Service, Hospital Universitario de Salamanca and IBSAL, Salamanca, Spain. mdiezcampelo@usal.es.
María
M
Diez-Campelo
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. angarmon@usal.es.
Andrés C
AC
García-Montero
Spanish Net on Aging and Frailty (RETICEF) Instituto de Salud Carlos III, Madrid, Spain. jfblanco@usal.es.|Orthopedics Service, Hospital Universitario de Salamanca and IBSAL, Salamanca, Spain. jfblanco@usal.es.
Juan F
JF
Blanco
Hematology Service, Hospital Universitario de Salamanca and IBSAL, Salamanca, Spain. concarol@usal.es.
Maria Consuelo
MC
Del Cañizo
Spanish Net on Aging and Frailty (RETICEF) Instituto de Salud Carlos III, Madrid, Spain. jpino@usal.es.|Rheumatology Service, Hospital Universitario de Salamanca and IBSAL, Salamanca, Spain. jpino@usal.es.
Javier
J
del Pino Montes
Department of Medicine and Cytometry Service (NUCLEUS), Cancer Research Center (IBMCC, USAL-CSIC), Institute for Biomedical Research of Salamanca (IBSAL), University of Salamanca (USAL), Salamanca, Spain. orfao@usal.es.|Centro de Investigación del Cáncer, Campus Miguel de Unamuno, 37007, Salamanca, Spain. orfao@usal.es.
Alberto
A
Orfao
D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D018826:CD13 Antigens / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation; D002478:Cells, Cultured; D000071063:Endoglin; D005260:Female; D006801:Humans; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism*; D008297:Male; D059630:Mesenchymal Stem Cells / Q000145:classification / Q000166:cytology* / Q000378:metabolism; D008875:Middle Aged; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*
D015703:Antigens, CD; C064206:ENG protein, human; D000071063:Endoglin; D011956:Receptors, Cell Surface; D017493:Leukocyte Common Antigens; D018826:CD13 Antigens