Mature accessory cells influence long-term growth of human hematopoietic progenitors on a murine stromal cell feeder layer.

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.

Impeded normal hematopoiesis in bone marrow of patients with multiple myeloma.

Insufficient output of mature blood cells frequently accompanies the typical impairments of late B cell development in multiple myeloma (MM). In a large group of previously untreated patients, bone marrow samples were analyzed and showed a general decrease of mononuclear cell (MNC) content. Colony growth of granulo-monocytic progenitors in short-term assays is compromised in a substantial number of patients at partly severe degrees, who at the same time show higher plasma cell content and belong to clinically more severe groups; the other patients show normal in vitro growth, contain less plasmocytes in the marrow and belong to varying degrees of aggressiveness. Thus a heterogeneity of the disease is emerging on the level of bone marrow cells which matches with high aggressiveness of the disease in one type. It can be speculated that in this type there are different underlying mutational events compared to the others: besides the characteristic changes in B cell differentiation, here the cellular defects have an impact on normal granulo-monocytic (and other) progenitor recruitment, which is absent in the other cases.

Isolation and identification of two CD34+ cell subpopulations from normal human peripheral blood.

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 CD10 (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+ high cells failed to establish colony growth in short-term culture while the larger and denser CD34+low 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 steady-state CD34+ cells from normal human peripheral blood. The smaller and less dense CD34+high cells correspond to the earliest progenitors that express differentiation markers poorly but CD34 antigen brightly, do not give rise to short-term colony growth in vitro, and thus represent indirect evidence for pluripotent hematopoietic stem cells (PHSC). The larger and denser CD34+low cells are the more mature progenitor cells, already committed to myeloid, lymphoid or erythroid differentiation but only dimly expressing CD34 antigen, and these cells were responsible for short-term colony growth in vitro.