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Role of Serum-Free Medium in the Ex Vivo Expansion of Human Cord Blood Hematopoietic Stem Cells

(summarized by Vivek Tanavde)

Dr. Richard Shadduck presented recent results on ex vivo expansion of cord blood stem/progenitor cells. He stressed the importance of using early acting cytokines like flt-3 ligand (FL) and thrombopoietin (TPO) in the ex vivo culture medium. Using "conventional" cytokines such as G-CSF, GM-CSF, and interleukin 3 (IL-3), other labs are able to achieve only limited expansion (two- to threefold) of cord blood CD34⁺ cells in vitro as quantified by flow cytometry. However using culture medium containing FL and TPO [1], the Shadduck lab was able to achieve much more impressive ex vivo expansion (20- to 30-fold) of cord blood CD34⁺ cells. Using this system, enough CD34⁺ cells could be generated from a single collection for potential engraftment of an adult (based on numbers of CD34⁺ cells/kg known to be required for engraftment). Addition of stem cell factor and IL-6 to the system further enhanced the numbers of CD34⁺ cells by two- to fourfold. Thus, 60- to 100-fold expansion of CD34⁺ cells from cord blood was observed by four to six weeks ex vivo culture.

They further studied the kinetics of stem cell expansion using this culture system. Using total cell count as a parameter in this study, a lag period of four to five weeks was observed when whole cord blood was used for expansion. A plateau in the growth curve was observed after four to five weeks when whole cord blood mononuclear cells were used. The lag was reduced to one week when purified CD34⁺ cells from cord blood were used. The frequency of the more primitive CD34⁺/CD38^dim population increased 10-fold in two months of culture using the same culture system.

Using serum free medium (QBSF-60) containing the above-mentioned cytokines (FL + TPO) for culture of CD34⁺ cells isolated from cord blood, the Shadduck lab observed that the cell numbers did not increase in the absence of serum. However, if cord blood CD34⁺ cells were cultured in serum-containing medium for an initial period of one to two weeks, the cultured cells could then be successfully transferred to QBSF-60 medium and expanded. Therefore, it appears that serum contains some uncharacterized growth factor(s) required for initiation of the CD34⁺ cell expansion in culture.

Thus, this ex vivo culture system has potential for clinical application for adult patients needing allogeneic stem cell transplants; ex vivo culture might be able to expand CD34⁺ cell numbers sufficiently to allow cord blood transplantation of large adult recipients. The Shadduck lab has recently developed a system using teflon bags for ex vivo culture of stem cells, and they are planning to perform long-term culture-initiating cell assays and in vivo engraftment assays to further evaluate potential stem/progenitor cell expansion in this system. Up to now, their emphasis has been on immunophenotyping of the ex vivo cultures without functional assay data. If encouraging results are obtained, cells could be transplanted into the patients directly from these cultures done in a closed system with minimum manipulation of the cells. The Shadduck lab is also attempting to identify the serum-derived factor(s) needed to enable initiation of primary cultures of cord blood CD34⁺ cells in serum-free medium.

References

1. Piacibello W, Sanavio F, Garetto L et al. Extensive amplification and self-renewal of human primitive hematopoietic stem cells from cord blood. Blood 1997;89:2644-2453.[Abstract/Free Full Text]

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