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Abstract. Circulating CD34' progenitors were separated from normal human peripheral blood on the basis of size and density by counterflow centrifugal elutriation (CCE). The CD34' cells, 0.15% of peripheral blood mononuclear cells, were heterogeneous with respect to their elutriation characteristics, mainly size and density. The least mature CD34' cells, characterized by lack of CD38 antigen, were predominantly found in the small lymphoid cell fraction. In fractions containing larger and denser cells (large lymphocytes, monocytes, and granulocytes), CD38 was increasingly expressed on the CD34+ cells, as were lineage commitment markers CDlO (B lymphoid), CD33 (myeloid), CD13 (myelomonocytic) and CD71 (erythroid) antigens. The smaller and less dense CD34+ cells expressed CD34 antigen brightly while the larger and denser CD34' cells expressed it dimly. The smaller and less dense CD34+b'gb cells failed to establish colony growth in short-term culture while the larger and denser CD34''"" cells gave rise to high counts of colony forming units-granulocyte macrophage (CFU-GM). Physical separation on the basis of size and density by CCE differentiates between two main classes of steadystate CD34' cells from normal human peripheral blood. The smaller and less dense CD34+h1gb cells correspond to the earliest progenitors that express differentiation markers poorly but CD34 antigen 'Present address Sir

We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay, now part of the new criteria of polycythemia vera.

A recently described long-term culture system for early human progenitor cells was established with the murine preadipocyte stromal line FBMD-1 grown in 96-well plates; cobblestone areas formed by inoculated hematopoietic cells are determined in a limiting dilution setting after five weeks' culture. To compare the capacity of cobblestone-area-forming cell (CAFC) formation by bone marrow and leukapheresis products in this system, mononuclear cells (MNC) of both origins were cultured. As related to CD34 + cell content, CAFC yields after five weeks' culture were in the same range in bone marrow and leukapheresis stemming from patients with efficient mobilization of hematopoietic cells. In purified CD34 + cell fractions, the CAFC yield per inoculated cell number was considerably higher than in MNC; however, if the CAFC number was related to the inoculated CD34 + cell number in MNC and after purification, the yield was four to eight times decreased in purified fractions. Addition of the mature cells brought the CAFC yield back up to the numbers obtained in the unseparated MNC fraction. By contrast, slightly more advanced progenitors per CAFC were found in cultures of purified hematopoietic cells from both origins than in whole MNC. The results suggest that mature human accessory cells give noticeable support to recruitment of early progenitors on this feeder but lead to lower yield of GM progenitors.

growth capacity of grafts is still CFU-GM content. The number of cells to be transplanted is adapted to the body weight of the patient and expressed as CFU-GM/kg. It is of concern, however, that there is no common agreement on the number of CFU-GM/kg actually required to ensure complete and stable hematopoietic recovery. While it is recommended that 5-10 times more CFU-GM/kg for peripheral stem cell grafts (PBSCT) than for autologous bone marrow grafts (ABSCT) be used, to compensate for the lower content of early stem cells in cytapheresis products, published values for both ABSCT and PBSCT vary by one to two orders of magnitude between different centers. This situation is irritating if clinical data, such as duration of aplasia after grafting, are to be compared between different centers and for different treatment regimes, but is even more confusing if centers without previous experience in transplantation search the literature for practical guidance.Two levels of uncertainty are the source of these differences: first, the methods for "stem cell" measurement vary between the laboratories. Second, in laboratory animals systematic experiments can be performed to determine the minimal dose of stem cells that is required for hematopoietic recovery; however, in patients it is more difficult to determine this dose, and to define what degree of maturation of cells is required. In fact, both of these shortcomings are closely entangled. To date, in each center, empirical numbers of CFU-GM (as grown in the locally used short-term assay) are transplanted, and this parameter indicates that the hematopoietic cells in the graft are within the safe range, both in terms of number and differentiation potential. To determine more information about actual minimal requirements for stem cells in grafts, precise and universally applicable parameters are needed, which allow correlation between numbers of grafted hematopoietic cells, their maturity at time of transplant, and the time to engraftment.Currently two different strategies are in use to achieve this goal. For the short-term assay, which measures the number of committed clonogenic progenitors, standard stem cell culture media have now become commercially available, permitting the use of identical conditions for both stimulation and colony growth. If counting of the colonies is then carried out using common evaluation criteria, accurate and comparable quantitation of the more mature stem cells (responsible for short-term recovery) should be possible in each center. This assay, however, still gives no information about less mature and nonclonogenic stem cells within grafts.Direct cytofluorimetric evaluation of immature cells provides new and promising potential in this regard. The total fraction of immature cells can be quantitated, since they all express the CD34 surface marker. However, this measurement of a minute cell fraction is technically demanding. Since both cytofluorimetry and the shortterm colony assay depend on the use of standardized procedures, materials, and eval...

Using three different statistical tests in parallel, we showed in a preliminary study that neither mononuclear cells, CD34+33+ or 33− cells, nor CD34+38+ cells significantly correlated with engraftment kinetics following autologous blood cell transplantation (ABCT). We additionally demonstrated here, in a series of patients suffering from malignant diseases, that the graft content in CD34+38− cells is individually a more sensitive indicator of the earliest, as well as the latest post‐ABCT trilineage hematopoietic recovery than the colony‐forming units‐granulocyte‐macrophage and even the total CD34+ cell content. This suggests that the CD34+38− cell population is itself subdivided into two more subsets, one being already lineage‐committed and responsible for short‐term engraftment, the other containing only very primitive hematopoietic cells responsible for sustained engraftment. Strong arguments favor the probability that these subsets correspond to HLA‐DR+ and DR− cells, respectively. We also defined an optimal threshold value of 0.05 × 106 CD34+38− cells/kg of the patient's body weight (b.w.) above which a rapid and sustained trilineage engraftment safely occurs. In fact, infusion of lower numbers of cells seems to have a more significant impact on long‐term compared to short‐term neutrophil recovery and on platelet kinetics engraftment. We additionally looked for the eventual influence on engraftment time of the type of disease, and of post‐ABCT administration of hematopoietic growth factors (HGF). When the type of disease appeared to have no influence on the engraftment time, posttransplant HGF administration significantly reduced the time to trilineage engraftment in patients transplanted with < 0.05 × 106 CD34+38+ cells, thus justifying it in case of reinfusion of low numbers of CD34+38+ cells. On the other hand, the administration of HGF after infusion of more than 0.05 × 106 CD34+38− cells/kg b.w. did not hasten more, or only very little, the engraftment time, thus becoming not only unprofitable for the patients but costly as well.

At the "2nd European Workshop on Stem Cell Methodology," held in Mulhouse, France, on May 3-7, 1993, part of the meeting was dedicated to the positive selection of CD34+ cells. All devices that are currently in use, or will be available in the near future, were explained and practically demonstrated using human cell populations by scientists involved in their development. In this paper, a review of these methods is given in the form of a short description, together with the data presented in Mulhouse and available from the literature.