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20. Multi-lineage expansion potential of primitive hematopoietic progenitors: superiority of umbilical cord blood compared to mobilized peripheral blood. Lewis ID, Verfaillie CM. Exp Hematol. 2000;28:1087-1095. The authors point out that the majority of studies assessing ex-vivo expansion of primitive hematopoietic cells only address production of myeloid progeny whereas it may be more appropriate to maintain or expand progenitors that retain capacity for multilineage differentiation. They assessed the capacity of the murine fetal liver cell line AFT024 to expand primitive myeloid progenitors (LTC-IC) and lymphoid progenitors (NK-IC) from umbilical cord blood (CB) and mobilized peripheral blood (PB) CD34(+)lin(-)38(-) cells. Sorted cells were established in expansion cultures in direct contact with the feeder or in a transwell above the feeder (noncontact culture) and various combinations of Flt-3L (FL), stem cell factor, interleukin 7, thrombopoietin (Tpo), and macrophage inflammatory protein-1alpha added. Frequency of LTC-IC and NK-IC was assessed at day 0 and following 2 and 5 weeks expansion culture. Cord blood contained significantly more LTC-IC and NK-IC at day 0 than PB blood and showed an enhanced capacity for expansion compared to PB. The combination of FL and Tpo showed maximal expansion of CB LTC-IC and NK-IC at 5 weeks in both contact and noncontact conditions. In contrast, expansion of PB LTC-IC and NK-IC was maximal at 2 weeks and required multiple cytokines. 21. A systematic strategy to optimize ex vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells. Yao CL, Chu IM, Hsieh TB, Hwang SM. Exp Hematol. 2004;32:720-7. The authors report a serum-free, stroma-free and chemically defined medium for hematopoietic stem cell (HSC) expansion. Mononuclear cells (MNCs) were isolated from umbilical cord blood. HSCs were stimulated to proliferate ex vivo in the MNC culture system with variable serum substitutes, cytokines, and basal media. The optimal compositions of serum substitutes and the cytokine cocktail for HSC expansion in the MNC culture system were BIT (4 g/L BSA, 0.71 microg/mL insulin, and 27.81 microg/mL transferrin), and CC-9 (5.53 ng/mL TPO, 2.03 ng/mL IL-3, 16 ng/mL SCF, 4.43 ng/mL FL, 2.36 ng/mL IL-6, 1.91 ng/mL G-CSF, 1.56 ng/mL GM-CSF, 2.64 ng/mL SCGF, and 0.69 ng/mL IL-11) in the Iscove's modified Dulbecco's medium. After 6-day culture, the absolute fold expansions for white blood cells, CD34+ cells, CD34+CD38- cells, CFC, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, 2.8-fold, respectively. The authors conclude that their formula had lower cytokine concentrations compared to others reported in the literature and commercial media, but had superior or comparable expansion ability on HSC growth. 22. In vitro and in vivo expansion of hematopoietic stem cells. Sauvageau G, Iscove NN, Humphries RK. Oncogene. 2004;23:7223-7232. This is a rather comprehensive review of expansion of hematopoietic stem cells. A variety of in vitro culture conditions have now been described that permit enormous expansion of later clonogenic progenitors and substantial expansion of even more primitive cells detected as long-term culture initiating cells. However, the in vitro expansion of rigorously defined HSC has proven a greater challenge. They cite encouraging evidence that net expansion of human and murine HSCs is possible in relatively simple, short-term liquid culture systems, but also indicate that the magnitude of expansion so far obtained is modest and dramatically less than the readily achievable multilog expansion of late progenitors. A recurring observation also is the inability to sustain the output of rigorously defined repopulating HSC beyond a very limited time in culture. Clues to extrinsic mediators of HSC self-renewal are also emerging from a broader understand of the key receptor signaling pathways involved in the development and maintenance of the hematopoietic system. The authors describe recent findings in this context that highlight promising additional avenues to HSC expansion. Hox genes are part of a highly conserved family of transcription factors that contains 39 members clustered on four different chromosomes. Experimental studies have led to four important conclusions: First, HSCs engineered to overexpress Hoxb4 were much more competitive in vivo than control cells, and this expansion had the signature of a potential effect on HSC self-renewal; second, the activity of Hoxb4 appeared cell autonomous since it was not transferred to untransduced cells within the same animal; third, the Hoxb4-induced HSC expansion is limited in time; fourth, Hoxb4-transduced cells seemed to respect the mechanisms regulating total HSC numbers in vivo. Several observations suggest that it might be possible to develop methods allowing the in vitro expansion of HSCs using direct delivery of a recombinant HOXB4 protein. The authors conclude that the remarkable capacity of specific HOX proteins to enhance self-renewal provides a powerful experimental handle for exploration of mechanisms and has potential clinical utility. 23. Clinical strategies for expansion of haematopoietic stem cells. Sorrentino BP. Nat Rev Immunol. 2004;4:878-88. In this detailed review, the authors review the molecular mechanisms that control HSC numbers and discusses how these can be modulated to increase the number of HSCs. Clinical applications of HSC expansion are then discussed for their potential to address the current limitations of HSC transplantation. They point out that the ability to increase the number of HSCs either in vivo or in vitro would provide new treatment options, but the amplification of HSCs has been difficult to achieve. Although many genes can influence HSC kinetics, this review focuses on those factors that show the greatest promise for controlling the amplification of HSCs in a clinical setting. These genes can be divided into three main categories: genes that control transcription factors such as homeobox B4 (HOXB4), genes that encode signaling molecules; and genes that regulate the cell cycle, such as p18 and p21. The authors extensively review available data and then discuss potential clinical applications, including the use of ex-vivo expanded HSCs in umbilical cord blood transplantation protocols. They conclude by stating that the ability to amplify HSCs remains one of the "holy grails" of hematology and bone marrow transplantation. This goal has remained elusive despite exhaustive attempts to define culture conditions that facilitate HSC expansion. However, recent discoveries regarding the molecular control of HSC self-renewal and cell division now place this goal within reach. 24. Ex vivo expansion of transplantable human hematopoietic stem cells: where do we stand in the year 2000? Shih CC, DiGiusto D, Forman SJ. J Hematother Stem Cell Res. 2000;9:621-628. Ex vivo expansion of hematopoietic precursors, progenitors and stem cells represents the modern era of cellular therapeutics in the 21st century. For the last 10 years, increasing means for identifying and purifying hematopoietic stem cells and cytokines have facilitated and improved the development of ex vivo stem cell expansion technology. However, technology has not yet reached a stage where ex vivo-expanded hematopoietic progenitors and stem cells can be used routinely for replacement therapy. Lessons learned over the past 10 years from investigations focused at developing optimal ex vivo stem cell expansion systems have continued to a much greater understanding of stem cell biology. This knowledge has led to novel attempts at ex vivo expansion of hematopoietic precursors, progenitors, and stem cells, and should facilitate development of a new generation of cellular therapeutics. This review addresses recent progress toward development of clinically useful protocols for stem cell expansion. In addition, we discuss the results of a limited number of clinical trials that address the efficacy of such procedures. Three major areas of ex vivo stem cell expansion that impact clinical feasibility are discussed, including: (1) selection of an optimal stem cell population for expansion, (2) definition of the desired characteristics of the expanded stem cell population to be used for engraftment, and (3) development of new reagents and procedures for expansion and infusion of hematopoietic progenitors and stem cells. 25. Ex vivo expansion of human umbilical cord blood and peripheral blood CD34(+) hematopoietic stem cells. Gilmore GL, DePasquale DK, Lister J, Shadduck RK. Exp Hematol. 2000;28:1297-1305. The authors examined the proliferation and expansion of human hematopoietic stem cells (HSC) in ex vivo culture with the goal of generating a suitable clinical protocol for expanding HSC for patient transplantation. HSC were derived from umbilical cord blood (UCB) and adult patient peripheral blood stem cell collections. HSC were stimulated to proliferate ex vivo by a combination of two growth factors, flt-3 ligand (FL) and thrombopoietin/c-mpl ligand (TPO/ML), and assessed for expansion by flow cytometry. Ex vivo expansion cultures of UCB were maintained for prolonged periods (up to 16 weeks), and sufficient HSC were generated for adult transplantation. In contrast to UCB, FL + TPO/ML did not significantly increase CD34(+) peripheral blood stem cell (PBSC) numbers. The authors concluded that UCB-HSC can be expanded in culture to numbers theoretically adequate for safe, rapid engraftment of adult patients. However, additional studies are needed to establish the functional activity of expanded UCB-HSC.
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Page Updated
16 Feb 2008 |
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