Use of CD9 expression to enrich for porcine hematopoietic progenitors.
OBJECTIVE
The aim of this study was to develop novel markers for enrichment of hematopoietic progenitors from bone marrow of swine.


METHODS
We previously showed that pig bone marrow contains a "side population" (SP) of Hoechst dye-effluxing cells that resembles the hematopoietic stem cell (HSC)-containing murine SP and therefore represents a putative pig stem cell population. We screened a panel of monoclonal antibodies for those that allowed positive or negative enrichment of porcine SP cells and tested one of these for enrichment of hematopoietic progenitors in short-term and long-term in vitro assays. We then screened an expression library to clone the gene whose product is recognized by this antibody.


RESULTS
Among a panel of 35 monoclonal lines screened, we found three that were useful for positive enrichment of SP cells and seven for negative enrichment. The 4-6 monoclonal line, allowing around 10-fold negative enrichment of SP cells, recognized the product of the porcine CD9 gene. Hematopoietic progenitors measured by short-term colony-forming unit and long-term cobblestone area-forming cell assays were around 10-fold enriched in the CD9(negative/low) fraction and were significantly depleted in the CD9(high) fraction.


CONCLUSIONS
The antibody against the porcine CD9 gene product may be of use for enrichment of porcine hematopoietic stem cells. This approach to identify novel markers for enrichment of hematopoietic progenitors may be applicable to other mammalian species.
Baylor College of Medicine, Center for Cell and Gene Therapy, Houston, TX 77098, USA.
Matthew
M
Heinz
Christene A
CA
Huang
David W
DW
Emery
Maria A
MA
Giovino
Annie
A
LeGuern
Barbara
B
Kurilla-Mahon
Pierre
P
Theodore
J Scott
JS
Arn
Megan
M
Sykes
Richard
R
Mulligan
Julian D
JD
Down
David H
DH
Sachs
Margaret A
MA
Goodell
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D001562:Benzimidazoles / Q000378:metabolism; D015415:Biomarkers; D001854:Bone Marrow Cells / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003114:Colony-Forming Units Assay; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D019650:Hematopoietic Stem Cell Mobilization / Q000662:veterinary; D006412:Hematopoietic Stem Cells / Q000737:chemistry / Q000145:classification / Q000378:metabolism; D006825:Hybridomas / Q000276:immunology; D008322:Mammals / Q000235:genetics; D008562:Membrane Glycoproteins; D051379:Mice; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013552:Swine; D013556:Swine, Miniature / Q000033:anatomy & histology*; D060245:Tetraspanin 29; D014162:Transfection
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D001562:Benzimidazoles; D015415:Biomarkers; C554870:Cd9 protein, mouse; D005456:Fluorescent Dyes; D008562:Membrane Glycoproteins; D060245:Tetraspanin 29; C017807:bisbenzimide ethoxide trihydrochloride