For further information on mesenchymal cells, see Annotated Bibliography, XIX. Miscellaneous Topics.

1. Ex vivo expansion of umbilical cord blood stem cells for transplantation: Growing knowledge from the hematopoietic niche. Hofmeister CC, Zhang J, Knight KL, Le P, Stiff PJ. Bone Marrow Transplant 2007;39:11-23.

This is a useful review for those interested in the past, present and possible future of ex vivo expansion of UCB stem cells for transplantation.

Initial efforts to expand UCB progenitors ex vivo have resulted in expansion of mature rather than immature HSC, confounded by the inability to accurately and reliably measure long-term reconstituting cells. Ex vivo expansion of UCB HSC has failed to improve engraftment because of the resulting defects that promote apoptosis, disrupt marrow homing and initiate cell cycling.

In this review, the authors discuss the future of ex vivo expansion after providing a review of early clinical experience, current needs for successful HSC expansion and recent attempts to optimize HSC culture conditions. They suggest that ex vivo expansion could be enhanced by manipulating newly discovered signaling pathways (Notch, Wnt, bone morphogenetic protein 4 and Tie2/angiopoietin-1) and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) in a manner that promotes HSC expansion with less differentiation.

Recent preclinical/clinical HSC expansion investigations are reviewed including transcription inhibition, copper chelation, and Wnt pathway activation. Finally, the authors discuss current/planned HSC expansion clinical trials, and conclude by stating that the ultimate clinical value of infusing expanded UCB is yet to be realized.

2. Ex vivo expansion does not alter the capacity of umbilical cord blood CD34+ cells to generate functional T lymphocytes and dendritic cells. Kobari L, Giarratana C, Gluckman JC, Douay L, Rosenzwajg M. Stem Cells. 2006;24:2150-7.

The authors examined whether ex vivo expansion of umbilical cord blood progenitor cells affected their capacity to generate immune cells such as T lymphocytes (TLs) and dendritic cells (DCs). The capacity to generate TLs from cord blood CD34(+) cells expanded for 14 days (d14) was compared with that of nonexpanded CD34(+) cells (d0) using fetal thymus organ cultures or transfer into nonobese diabetic/severe combined immunodeficient mice.

The cell preparations yielded comparable percentages of immature (CD4(+)CD8(-), CD4(+)CD8(+)) TLs and functional mature (CD3(+)CD4(+), CD3(+)CD8(+)) TLs with an analogous TCR (T-cell receptor)-Vbeta repertoire pattern. As regards DCs, d0 and d14 CD34(+) cells also yielded similar percentages of CD1a(+) DCs with the same expression levels of HLA-DR, costimulatory and adhesion molecules, and chemokine receptors. DCs derived from either d14 or d0 CD34(+) stimulated allogeneic TLs to the same extent, and the cytokine pattern production of these allogeneic TLs was similar with no shift toward a predominant Th1 or Th2 response.

Even though the intrinsic capacity of d14 CD34(+) cells to generate DCs was 13-fold lower than that of d0 CD34(+) cells, this reduction was offset by the prior amplification of the CD34(+) cells, resulting in the overall production of 15-fold more DCs.

The authors conclude that their data indicate that ex vivo expansion of CD34(+) cells does not impair T lymphopoiesis nor DC differentiation capacity.

3. Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation. Introna M, Franceschetti M, Ciocca A, Borleri G, Conti E, Golay J, Rambaldi A. Bone Marrow Transplant. 2006; 38:621-7.

CB transplantation is gaining increasing attention for the several advantages that it can offer with respect to the traditional adult BM transplant. However, one major disadvantage lies in the unavailability of the donor for patients experiencing graft failures nor of DLI for recipients who suffer leukemia relapse.

Cytokine-induced killer (CIK) cells are naturally occurring cytotoxic cells active against a variety of leukemia and lymphoma targets and with low or absent activity against normal BM stem cells and tissues. Their in vitro expansion is easily obtained applying standardized protocol which can be utilized under strict adherence in GMP conditions, as already has been established for the autologous setting. This paper presents the idea that a salvage CIK infusion may be potentially planned for patients experiencing leukemia relapse after CB transplantation. Indeed, the authors demonstrated that simply washing the CB unit bag at the end of the infusion is sufficient to recover a number of functionally active CIK cells, which could be infused.

The authors used a standardized 21-day expansion protocol to produce CIK cells starting from very small amounts of nucleated cells (approximately 15 x 10(6) cells) isolated from cord blood.

Mononuclear cells are stimulated with anti CD3 (OKT3) and IFNγ and then expanded with IL-2. Washouts of cord blood units bags (at the end of the infusion) may be sufficient to yield almost 500 x 10(6) CIK by the same expansion protocol.

CIK cells show strong cytotoxic activity against a variety of tumor target cell lines including B and T lymphomas and myeloid leukemias. More importantly, expanded cord blood-derived CIK cells are cytotoxic against fresh leukemic blasts and express perforin, granzyme and NKG2D molecule at high levels.

The same in vitro protocol has already been used to expand CIK cells from peripheral blood of adult donors under GMP conditions. CIK cells have been used in a phase I study at 10 x 10⁶/kg or above in the autologous setting. The present observations open up the possibility of imagining a future clinical application of leukemia relapse following cord blood transplantation with CIK cells obtained from the same cord blood unit.

4. Ex vivo expansion of umbilical cord blood stem cells for transplantation: growing knowledge from the hematopoietic niche. Hofmeister CC, Zhang J, Knight L, Le P, Stiff PJ. Bone Marrow Transplant. 2007;39:11-23.

Initial efforts to expand UCB progenitors ex vivo have resulted in expansion of mature rather than immature HSC, confounded by the inability to accurately and reliably measure long-term reconstituting cells. Ex vivo expansion of UCB HSC has failed to improve engraftment because of resulting defects that promote apoptosis, disrupt marrow homing and initiate cell cycling.

In this rather detailed review the authors discuss the future of ex vivo expansion, which they suggest will include the isolation of immature hematopoietic progenitors on the basis of function rather than surface phenotype and will employ both cytokines and stroma to maintain and expand the stem cell niche. They suggest that ex vivo expansion could be enhanced by manipulating newly discovered signaling pathways (Notch, Wnt, bone morphogenetic protein 4 and Tie2/angiopoietin-1) and intracellular mediators (phosphatase and tensin homolog and glycogen synthase kinase-3) in a manner that promotes HSC expansion with less differentiation.

The authors predict that improved methods for ex vivo expansion will make UCBT available to more patients, decrease engraftment times and allow more rapid immune reconstitution post transplant.

5. International Forum: 1 Engelfriet, C. P. & Reesink., H. W. Vox Sanguinis 2005;89:172-173.

This is an International Forum regarding ex vivo expansion of hematopoietic precursor cells (HPCs). The authors of the Forum state that there are several interesting questions related to ex vivo expansion. For example, there seems to be a danger that expansion culture may be harmful to HPCs and there is the question of whether CD34+ cells should be isolated for expansion.

The authors posed 6 questions to 4 experts. The questions were:

Question 1. Do you agree that there is a danger that expansion culture may be harmful to haematopoietic stem cells? In view of this problem, if you practice the ex vivo expansion of HPCs:

• Do you use a fraction of a CB unit for expansion, infusing the remainder unmanipulated? If so, what is the time-span between infusion of the latter and that of the expanded cells?
• Do you use a fraction of a CB unit for expansion, infusing the remainder unmanipulated? If so, what is the time-span between infusion of the latter and that of the expanded cells?
• Do you use a fraction of a CB unit for expansion, infusing the remainder unmanipulated? If so, what is the time-span between infusion of the latter and that of the expanded cells?

Question 2. Do you isolate CD34⁺ cells for expansion? If so, which techniques do you use for isolating the CD34⁺ cells?

Question 3. Which growth factors do you use for expansion? And for what reason?

Question 4. Do you think that single or different cells are responsible for neutrophil-platelet and red cell expansion?

Question 5. Do you think that adding mesenchymal cells or intrabone transfusion facilitates in vivo engraftment?

Question 6. Do you think that infusing two unmanipulated CB units solves the problem? It has been reported that in that case, ultimately only one of the two CB units gives long-term engraftment. Have you any experience concerning this problem?

The experts who responded to the survey were:

• E Dickmeiss, Copenhagen, Denmark
• L Lazzarri, T Montemurro, and P. Rebulla, Milan, Italy
• Joan Garcia, Barcelona, Spain
• J. Wagner, Minneapolis, Minnesota, USA

A summary of the responses of the experts, which was to a large extent supplied by the authors of the Forum, is as follows:

Although there are no further actual data in the answers to show that ex vivo expansion may be harmful to HPCs, in those centers where expansion is practiced, protocols have been adopted that take this possibility into account: either only part of the CB unit is used for expansion, or an unexpanded unit is co-infused with an expanded unit. The Danish group do not practice ex vivo expansion because, to their knowledge, it is not possible to expand primitive HPCs ex vivo and they do not find expansion of short-term repopulating committed HPCs to the expense of the primitive HPCs a viable solution for the problem.

CD34⁺ cells are, in fact, isolated for expansion in Italy and Spain by using the CliniMACS from Miltenyi Biotech (Glodbach, Germany). In Minnesota, this selection step is being evaluated.

In contrast to the opinion of the Danish experts, the Italian and Spanish groups are of the opinion that an optimal combination of growth factors leads to the expansion of primitive HPCs.

Because primitive HPCs are expanded, it is postulated that all cell lineages are derived from a single expanded primitive HPC.

Some recently published data indicate that culture of CB stem cells on a mesenchymal all-feeding layer supports the ex vivo expansion of HPCs and that the co-transfusion of CB and mesenchymal cells may facilitate in vivo engraftment. In Spain, mesenchymal cells are co-transfused, especially in cases in which the micro-environment has suffered through treatment regimes.

Dr. Wagner cited published data on double cord blood transplants and stated that, to date, 37 such transplants have been performed after myeloablative conditioning at the University of Minnesota. All of the patients engrafted at a median of 23 days. Although he states that he believes that treatment with cyclophosphamide, fludarabine and TBI (1320 cGy) followed by double cord blood transplant has addressed the issue of engraftment and universal availability of umbilical cord blood for adult patients, the question of whether the speed of engraftment can be improved by this approach remains to be proven.

The authors of the Forum concluded that, although the ex vivo expansion of CB HPCs is now obviously routinely practiced in some centers, questions concerning important aspects of this procedure remain. "Perhaps we have endeavored to approach this subject at too early a stage, as several aspects of the procedure are still in the experimental phase."

6. Direct evidence for ex vivo expansion of human hematopoietic stem cells. Ando K, Yahata T, Sato T, Miyatake H, Matsuzawa H, Oki M, Miyoshi H, Tsuji T, Kato S, Hotta T. Blood. 2006;107:3371-7.

Over the past 15 years many investigators have undertaken experiments to study ex vivo expansion of HSCs. These studies have led to a number of clinical trials to evaluate ex vivo-expanded cells in patients. However, no significant clinical benefit has been demonstrated to date.

To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done.

The authors used lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells to provide the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro.

The authors conclude that their data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion, and the clonal tracking employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

7. Ex vivo expansion of umbilical cord blood. Robinson S, Niu T, de Lima M, Ng J, Yang H, McMannis J, Karandish S, Sadeghi T, Fu P, del Angel M, O'Connor S, Champlin R, Shpall E. Cytotherapy. 2005;7:243-50.

To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted.

Ex vivo expansion can be performed on whole CB units or a fraction of a CB unit that is combined with its unmanipulated fraction at the time of transplantation, or transplanted a period of time after the unmanipulated fraction. Clinical protocols that explore these approaches are currently under way at a number of medical centers.

Currently, ex vivo expansion processes include: (1) liquid expansion: CD34⁺ or CD133⁺ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed in this review and preliminary data that are available are provided.

Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.

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