Clinical isolation and functional characterization of cord blood CD133+ hematopoietic progenitor cells.
BACKGROUND
Human cord blood is a relevant source of CD133+ HPCs. Clinical-scale isolation of human umbilical cord blood (UCB) CD133+ HPCs using immunomagnetic microbeads and the CliniMACS clinical cell isolator is reported. CD133+ HPCs isolated after large-scale processing were functionally characterized.


METHODS
Closed disposable sets were used to process nine different samples of RBC-reduced UCB nucleated cells. In-vitro hematopoietic assays and human xenografts in NOD/SCID mice were performed to assess the functional properties of isolated CD133+ cells. Different mixtures of human cytokines were tested for the ability to expand nascent CD133+ HPCs. Furthermore, freshly isolated CD133+ cells were conditioned in culture medium specifically tested to support in-vitro myogenesis or osteogenesis.


RESULTS
Isolation procedures yielded the recovery of an average of 2.53 x 10(6) CD133+ HPCs with a mean recovery of 96 percent (referred to as RBC-reduced samples) and a final sample purity of 82 percent. Purified CD133+ cells had high cloning efficiency, had relevant long-term activity, and were capable of repopulating irradiated NOD/SCID mice. In 10-day stroma-free cultures, a 2-fold and 8.3-fold expansion of colony-forming cells (CFCs) and extended long-term culture-initiating cells, respectively, was obtained. Freshly isolated CD133+ cells differentiated into large nucleated cells expressing either myosin D or osteopontin (as revealed by RT-PCR and immuno-cytochemistry), with a protein/mRNA expression comparable to or even higher than that observed in UCB CD133- nucleated cells in identical culture conditions.


CONCLUSIONS
Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primitive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.
Bonanno
Giuseppina
G
Department of Gynecology and Obstetrics, and UNICATT Cord Blood Bank, Catholic University Medical School, Rome, Italy.
Perillo
Alessandro
A
Rutella
Sergio
S
De Ritis
Daniela Giovanna
DG
Mariotti
Andrea
A
Marone
Maria
M
Meoni
Franco
F
Scambia
Giovanni
G
Leone
Giuseppe
G
Mancuso
Salvatore
S
Pierelli
Luca
L
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D005312:Fetal Blood / Q000166:cytology*; D006023:Glycoproteins / Q000097:blood*; D006410:Hematopoiesis; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D024510:Muscle Development; D010012:Osteogenesis; D010455:Peptides / Q000097:blood*
D000071916:AC133 Antigen; D015703:Antigens, CD; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse