Pergamon

Transfus. Sci. Vol. 17, No. 2, pp. 291-293, 1996 Copyright © 1996 Elsevier Science Ltd. All rights reserved

PII:S0955-3886(96)00018-5 Printed in Great Britain 0955-3886/96 $15.00 + .00

Stem Cells: What's Happening? M. J. S e g h a t c h i a n , P h D

• Stem cell enthusiasts are probably aware that we are experiencing an exciting period of new development in the field of progenitor cells. This brief report focuses on selected topics which have been recently reported in two special interest group meetings in the U.K. The potential advantage of stem cells derived from human umbilical cord (HUC) blood over normal bone marrow (NBM) or peripheral blood stern cells (PBSC), as source products for reconstitution of the immune system is briefly mentioned, together with some potential applications of HUC blood in gene therapy. Copyright © 1996 Elsevier Science Ltd •

THE CHARACTERISTIC PROPERTIES AND STANDARDISATION ASPECTS OF CD34 SURFACE ANTIGEN

The value of CD34 positive testing in haematopoiesis and the assessment of the char- acteristic properties of stem cells is now well established. CD34 is a cell surface antigen selectively expressed on human haematopoietic progenitor cells and its determination is of value for the enumeration, isolation and manipulation of these cells. Struc- turally, CD34 appears to have a rod-like conformation and consists of a core protein backbone of 40 kDa with extensive glycosylation and sialylation. The development of monoclonal antibodies to CD34 has been extremely useful in the identification of var- ious cells which express CD34 (i.e., vascular endothelium, lymphatic high endothelial venules and some fibroblasts, etc). These monoclonal antibodies are currently classified into three groups depending on their enzymatic sensitivities and several other characteristic properties of various epitopes. Not all haematopoietic cells have identical epitope expression and some appear to be differently glycosylated. The sialo- mucin structure of vascular endothelial cells rapidly downregulates during vascular inflammatory reactions, suggesting that CD34 may have a role in modulating cellular adhesion (i.e., promoting adhesion to bone marrow stroma) but the true nature of CD34 biological function remains to be fully elucidated. Although the assessment of the CD34 positive cells is essential for both selection and ex vivo expansion of stem cells, nevertheless we do not yet have a proper quality assessment and standardised method for CD34 positive cell testing, to enable us to effectively compare data and/or recommend an accepted standard procedure for CD34 ÷ enumeration. This remark is pertinent to the evaluation of the total and differential cell counting of stem cell prod- ucts, as well as to the determination of an accepted standard for CD34" enumeration by either flow cytometry or clonogenic assays. A fully fledged standardisation pro- gramme at the national level is an extremely important step and one which, I am sure, will be enthusiastically pursued.

North London Blood Transfusion Centre, Colindale Avenue, London NW9 5BG, U.K.

291

292 Transfus. Sci. Vol. 17, No. 2

THE MOBILISATION AND POSITIVE SELECTION OF HAEMATOPOIETIC PROGENITOR CELLS

Pluripotent haematopoietic stem cells circulate in the peripheral blood; during the early recovery phase of myelosuppressive chemotherapy large numbers appear in the circulation. Cytokines, such as G-CSF, in combination with myelosuppressive chemotherapy, are used to increase the circulation levels of haemopoietic cells. This approach allows the reduction of both drug dosage and morbidity/mortality rates associated with high dose drugs such as cyclophosphamide. Apheresis procedures are used for the collection of stem cells for the purpose of auto-transplants after drug therapy. Again, by monitoring the recovery of leucocytes and CD34 + cells in the circulation, it is possible to optimise the time for collection. This is now often only one or two procedures. Concern is nevertheless expressed that peripheral blood stem cells (PBSC) harvests may also contain tumour cells, requiring investigation of residual disease and contam- ination of harvest. Purging strategies may prove justified, where significant residual contamination of CD34+ cells exists. This is easily achieved by using immunoaffinity selection (i.e., with the Cellpro as a purging technique) without affecting the clinical effec- tiveness of the purified products. The use of such a product is associated with rapid haemopoietic engraftment, particularly of platelets. This contributes both to a reduction in time spent in hospital and to a reduction of the pre-transplant morbidity/mortality rates.

H U M A N UMBILICAL CORD (HUC) BLOOD VERSUS NBM

The fact that HUC blood is readily available and contains high levels of lymphocytes, with a rather naive phenotype and with the additional advantages of crossing HLA barriers, has led to rapid expansion of development work on areas of optimisation of collection, manipulation and cryopreservation procedures. To date, it is well estab- lished that HUC blood provides an alternative supplementary source of unrelated HLA matched stem cells for transplantation in paediatric recipients; sustained grafts occurring in the majority of cases. Caution was expressed on the question of whether a single HUC blood donation is sufficient to engraft an adult recipient, as most of earlier successful transplantations were performed in patients <30 kg. Nevertheless, several scientific breakthroughs, using in vitro and ex vivo expansion of the stem cells in allogeneic transplantation, autologous support, syngeneic support and allogeneic unrelated support have been now described, justifying the potential role of HUC blood in both adults' transplantation and gene therapy. This is supported by the recent report that although in the clonogenic assay, HUC blood from the full-term baby contains an average 1.6 (+1) x 109 nucleated cells with 1.1 (+0.8) x 106 CFU-Mix, CFU-Meg, BFU-E and CFU-GM, which is in the lower range of reported values to engraft adult recipients of siblings with normal bone marrow (NBM). In long term culture, however, the HUC blood consistently shows large primitive colonies which proliferate and live for a significantly longer period than NBM (P = 0.0007). Accordingly, HUC blood appears to generate a higher proportion of haemopoietic cells as compared with NBM, and these may give sustained engraftment in adult recipients as well. It is noteworthy that the level of patient rejection of cord blood grafts is significantly lower than with bone marrow. Thus, hopefully greater donor mismatches could be tolerated without potential risk.

GENE THERAPY WITH CORD BLOOD

In respect of gene therapy, the use of retroviral and adeno-associated viral vectors to place genes for haematopoietically active cytokines and their receptors into either

Stem Cells: What's Happening? 293

fresh or cryopreserved stem and progenitor cells from cord blood, as possible therapeu- tic support, has now become established. Moreover, gene transduction and transplan- tation of HUC blood CD34 ÷ cells from ADA-deficient SCID and preclinical modeling of stem cell-based HIV gene, as a possible therapeutic modality for the treatment of AIDS are two good examples for further development of this rapidly expanding field of science.

FUTURE TRENDS

Despite some additional bonuses of HUC blood compared to NBM or PBSC, there are still significant operational, organisational and legal/ethical/regulatory issues that must be clarified. Similarly, methods for optimal expansion, the extension of transplants to adults and clinical experience with leukaemia or lymphoma patients need to be fully explored. Similarly, appropriate quality assurance schemes for collection/processing- induced "storage lesion" of such products need to be established. This is only the beginning of definitive demonstration of the benefit of various strategies for trans- plantation will come after careful assessment of efficacy and cost/benefit ratio, before the full potential of HUC blood in specific vs NBM or PBSC is fully realised. Nevertheless there are several important questions, in respect to the 3Ps of quality (patients, process, product), that need to be urgently addressed:

(i) Is HUC blood equivalent or truly superior to bone marrow in transplant?

(ii) What is the measurable clinical end-point?

(iii) What other variables and considerations are needed to be taken into account for the way forward and the future plan?

Much has been achieved and much more remains to be elucidated in this exciting field, justifying the existence of such a title as "What is Happening on Stem Cells?" as a regular feature of Transfusion Science.