Characterization of Serum-Free Ex Vivo–Expanded Hematopoietic Stem Cells Derived from Human Umbilical Cord Blood CD133⁺Cells

Abstract

The development of ex vivo expansion of hematopoietic stem cells (HSCs) is a promising approach to restore the required bone marrow function of patients with hematological disorders. Previously, we have reported the development of an optimized serum-free and cytokines-limited defined medium using statistic methodology for umbilical cord blood-derived HSC expansion. The aim of this study was to analyze further the characteristics and functions of cells in vitro and in vivo when cultured in this defined medium. After a 7-day batch culture, the average absolute fold expansions for CD133(+) cells, CD34(+)CD133(+) cells, CD34(+)CD38() cells, CD133(+)CD38(-) cells, CD34(+)CXCR4(+) cells, CD133(+)CXCR4(+) cells, and long-term culture-initiating cells were 21-, 20-, 723-, 618-, 160-, 384-, and 8-fold, respectively. The high enrichment of CD38(-) cells and CXCR4(+) cells of the CD34(+) subpopulation provided a very early uncommitted HSC proliferation and homing ability. Furthermore, the expanded cells showed a high level of telomerase activity to maintain their telomere length and repopulated the lethally irradiated NOD/SCID mice in vivo. These results indicated that the cytokines limited expanded cells from CD133(+) cells could substantially support simultaneous expansion of various stem/progenitor cells and engraft with the expanded cells from a low number of HSCs initially.

Protein-arginine Methyltransferase 1 Suppresses Megakaryocytic Differentiation via Modulation of the p38 MAPK Pathway in K562 Cells

“…Department of Health, Taiwan) and after approval from the scientific committees of the Food Industry Research and Development Institute, Taiwan. Isolation and expansion of CD34 ϩ cells were performed as described previously (28). Briefly, the CD34 ϩ cells were purified with CD34 microbeads by a Miltenyi VarioMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultivated in serum-free Iscove's modified Dulbecco's medium (HyClone) supplemented with serum substitutes (1.5 g/liter bovine serum albumin, 4.4 g/ml insulin, 60 g/ml transferrin, and 25.9 M 2-mercaptoethanol) and a cytokine mixture (8.5 ng/ml TPO, 4.1 ng/ml interleukin-3, 15 ng/ml stem cell factor, 6.7 ng/ml flk2/flt3 ligand, 0.8 ng/ml interleukin-6, 3.2 ng/ml granulocyte colony-stimulating factor, and 1.3 ng/ml granulocyte-macrophage colony-stimulating factor) for 6 to 7 days.…”

Engineering cord blood to improve engraftment after cord blood transplant

“…Multiple methods of CB expansion have been testedvarying remarkably from the starting cell population used for expansion (CD34 + versus CD133 + selected), the type of device used for cell extraction (Miltenyi CliniMacs or Nexell Isolex-300i), type of culture media (with or without serum), combination of cytokines [stem cell factor (SCF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), Fms-like tyrosine kinase 3 ligand (FLT-3L), IL-3, IL-6, IL-11, megakaryocyte growth and differentiation factor (MGDF) and thrombopoietin (TPO)], duration of culture (ranging from 1-10 weeks) and whether or not stromal support is used (31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). In addition, the timing of thawing of the CB unit in context of the timing of transplant, and the proportion of CB unit used for expansion also varied considerably.…”

Human Umbilical Cord Blood Stem Cells Rescue Ischemic Tissues

“…Unfortunately, current methods of expanding HUCB cells do not preserve the quality of the hematopoietic progenitor cells through to the end product (Jaroscak et al, 2003;Robinson et al, 2005). Current expansion methods cannot make up for the cells lost in the storage process, let alone augment them to a suitable dosing regimen (Hows et al, 1992;Liu et al, 2006;Traycoff et al, 1995;Yao et al, 2006). However, in the last couple of years, there have been several studies looking at ways to maximize yield, storage and recovery, and expansion which require validation (Gonzalez et al, 2010;Koestenbauer et al, 2009;Lin et al, 2010;Song et al, 2010;Vanneaux et al, 2010;Zeisburger et al, 2010).…”