title: Clinically relevant expansion of hematopoietic stem cells with conserved function in a single-use, closed-system bioprocess.
abstract: The clinical potential of umbilical cord blood-derived stem and progenitor cells has been demonstrated in various animal and human transplantation studies. However, the need for increased numbers of appropriate umbilical cord blood-derived cells continues to limit the development and success of these therapies. Ex vivo expansion has been widely studied as a method to overcome this limitation. We describe the use of a clinically relevant single-use, closed-system bioprocess capable of generating greater numbers of hematopoietic stem and progenitor cells that maintain in vivo and in vitro developmental potential. In addition to expanded numbers of CD34+ cells, CD34(+)CD38(-) cells, colony-forming cells, and long-term culture-initiating cells, the bioprocess generated > or =3.3-fold more long-term nonobese diabetic/severe combined immunodeficient repopulating cells (quantitatively determined using limiting dilution analysis) than present at input. Interestingly, these cells were also capable of multilineage engraftment and were shown to maintain their engraftment potency on a per long-term nonobese diabetic/severe combined immunodeficient repopulating cell basis compared with input noncultured cells. The developmental capacity of bioprocess-generated cells was further demonstrated by their ability to repopulate secondary nonobese diabetic/severe combined immunodeficient recipients. In vitro lineage analysis confirmed that bioprocess-generated cells could differentiate into myeloid and natural killer, B, and T cell lymphoid lineages. This in-depth analysis describes a bioprocess that generates human hematopoietic stem and progenitor cells with conserved hematopoietic activity, establishes analysis criteria for in vitro hematopoietic stem cell expansion studies, and serves as a foundation to test the therapeutic utility of cultured hematopoietic stem cells in large animals and humans.
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Madlambayan
authors.forename: Ian
authors.initials: I
authors.lastname: Rogers
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Purpura
authors.forename: Caryn
authors.initials: C
authors.lastname: Ito
authors.forename: Mei
authors.initials: M
authors.lastname: Yu
authors.forename: Daniel
authors.initials: D
authors.lastname: Kirouac
authors.forename: Robert F
authors.initials: RF
authors.lastname: Casper
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
mesh_terms: D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation; D019070:Cell Lineage; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured / Q000166:cytology; D003114:Colony-Forming Units Assay; D036101:Cord Blood Stem Cell Transplantation / Q000379:methods; D005312:Fetal Blood / Q000166:cytology*; D006085:Graft Survival; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D016130:Immunophenotyping; D007231:Infant, Newborn; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D011828:Radiation Chimera; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D014183:Transplantation, Heterologous