Stefan Scheding

Expression of Novel Surface Antigens on Early Hematopoietic Cellsa

Abstract: The purpose of this report is to demonstrate the expression of very recently identified surface antigens on CD34+ and AC133+ bone marrow (BM) cells. Coexpression analysis of AC133 and defined antigens on CD34+ BM cells revealed that the majority of the CD164+, CD135+, CD117+, CD38low, CD33+, and CD71+ cells resides in the AC133+ population. In contrast, most of the CD10+ and CD19+ B cell progenitors and a fraction of the CD71high population are AC133−, indicating that CD34+AC133+ cells are enriched in primitive and myeloid progenitor cells, whereas CD34+AC133− cells mainly consist of B cell and late erythroid progenitors. This corresponds to the highly reduced percentage of CD10+ B cells and the absence of CD71high erythroid progenitors on AC133+ selected BM cells. A portion of 0.2‐0.7% of the AC133+ selected cells do not coexpress CD34. These cells are very small and define a uniform CD71−, CD117−, CD10−, CD38low, CD135+, HLA‐DRhigh, CD45+ population with unknown delineation. Four color analysis on CD34+CD38− BM cells revealed that virtually all of these primitive cells express AC133. Using an improved liposome‐enhanced labeling technique for the staining of weakly expressed antigens, subsets of this population could be identified which express the angiopoietin receptors TIE (67.6%) and TEK (36.8%), the vascular endothelial growth factor receptors FLT1 (7%), FLT4 (3.2%), and KDR (10.4%), or the receptor tyrosine kinases HER‐2 (15.4%) and FLT3 (CD135; 77.6%). Our results suggest that the CD34+CD38− population is heterogeneous with respect to the expression of the analyzed receptor tyrosine kinases.

Approaches to Dendritic Cell‐Based Immunotherapy after Peripheral Blood Stem Cell Transplantation

Abstract: High‐Dose chemotherapy with peripheral blood progenitor cell transplantation (PBPCT) is a potentially curative treatment option for patients with both hematological malignancies and solid tumors, including breast cancer. However, based on a number of clinical studies, there is strong evidence that minimal residual disease (MRD) persists after high‐dose chemotherapy in a number of patients, which eventually results in disease recurrence. Therefore, several approaches to the treatment of mrd are currently being evaluated, including treatment with dendritic cell (DC)‐based cancer vaccines. DCs, which play a crucial role with regard to the initiation of T‐lymphocyte responses, can be generated ex vivo either from CD34+ hematopoietic progenitor cells or from blood monocytes. They can be pulsed in vitro with tumor‐derived peptides or proteins, and then used as a professional antigen‐presenting cell (APC) vaccine for the induction of antigen‐specific T‐lymphocytes in vivo. This paper summarizes our preclinical studies on the induction of primary HER‐2/neu specific cytotoxic T‐lymphocyte (CTL) responses using peptide‐pulsed DC. As HER‐2/neu is overexpressed on 30‐40% of breast and ovarian cancer cells, this novel vaccination approach might be particularly applicable to advanced breast or ovarian cancer patients after high‐dose chemotherapy and autologous PBPCT.

How many myeloid post-progenitor cells have to be transplanted to completely abrogate neutropenia after peripheral blood progenitor cell transplantation?: Results of a computer simulation

Although hematopoietic recovery following high-dose chemotherapy (HD-CT) and peripheral blood progenitor cell (PBPC) transplantation is rapid, there is still a 5- to 7-day period of severe neutropenia which, theoretically, might be abrogated by an additional transplantation of more differentiated myeloid post-progenitor cells (MPPC). However, both the number of MPPC required to abrogate neutropenia as well as the optimum scheduling of MPPC infusions are currently unknown. Therefore, these questions were addressed by applying a computer model of human granulopoiesis. First, model calculations simulating varying levels of chemotherapy dose intensity were performed and compared with typical clinical neutrophil recovery curves. Using this approach, the data for HD-CT without PBPC transplantation could be reproduced by assuming a reduction of stem cells, committed granulopoietic progenitors and proliferating precursors to about 0.001% of normal. PBPC-supported HD-CT was reproduced by increasing the starting values to at least 0.1%, which corresponded to about 1 to 2 x 10(5)/kg transplanted CFU-GM. Interestingly, reproduction of PBPC-supported HD-CT data could be observed for a wide range of starting values (0.1%-10% of normal), thus confirming the clinical observation that hematopoietic recovery after PBPCT cannot be improved by increasing the dose of transplanted cells over a certain threshold. Using the same simulation model, we then studied the effects of an additional MPPC transplantation. The results showed, that at least 5.7 X 10(8) MPPC/kg have to be provided in addition to the normal PBPC graft to avoid neutropenia <100/microL, and that MPPC are best transplanted on days 0 and 6 after HD-CT. Assuming a 100- to 120-fold cellular ex-vivo expansion rate and MPPC representing about 70% of total expanded cells, 5.7 X 10(8) MPPC/kg could be generated starting from 1 to 2 leukapheresis preparations with about 7 to 8 x 10(6) CD34+ PBPC/kg. Considering furthermore, that only a fraction of ex-vivo generated cells will seed and effectively produce neutrophils in-vivo, the required number of MPPC is most likely even higher and, therefore, might be difficult to be achieved clinically. However, the validity of the model results remains to be proven in appropriate clinical studies.

Effective ex vivo generation of granulopoietic postprogenitor cells from mobilized peripheral blood CD34+ cells

OBJECTIVE Neutropenia following high-dose chemotherapy and peripheral blood progenitor cell (PBPC) transplantation might be abrogated by an additional transplantation of ex vivo generated granulopoietic postprogenitor cells (GPPC). Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for optimum GPPC production. MATERIALS AND METHODS CD34(+) PBPC were cultured in serum-free medium comparing different (n = 32) combinations of stem cell factor (S), interleukin 1 (1), interleukin 3 (IL-3) (3), interleukin-6 (6), erythropoietin (E), granulocyte colony-stimulating factor (G), granulate-macrophage colony-stimulating factor (GM), daniplestim (D, a novel IL-3 receptor agonist), and Flt3 ligand (FL) under various culture conditions. Ex vivo generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. RESULTS Addition of G +/- GM but not GM alone to cultures stimulated with S163E effectively induced the generation of GPPC. GPPC production was maximum after 12 to 14 days. Best expansion rates were observed when cells were cultured at 1.5x10(4)/mL in 21% O(2). Modifications of culture conditions were either less or equally effective (i.e., modification of starting cell concentrations, low oxygen, addition of serum albumin or autologous plasma, repetitive feeding). Comparison of different cytokine combinations revealed that the optimum GPPC expansion cocktail consisted of S6GD+FL (day 12: 130-fold cellular expansion, 32% myeloblasts/promyelocytes, 49.4% myelocytes/metamyelocytes, 12.4% bands/segmented), which furthermore expanded CD34(+) cells (3.4-fold) and clonogenic progenitors (13.4-fold). CONCLUSION Using the S6DG+FL expansion cocktail, GPPC could be effectively produced ex vivo starting from positively selected CD34 PBPC, possibly enabling amelioration or even abrogation of posttransplant neutropenia.

Effective ex vivo generation of megakaryocytic cells from mobilized peripheral blood CD34+ cells with stem cell factor and promegapoietin

OBJECTIVE The additional transplantation of ex vivo-generated megakaryocytic cells might enable the clinician to ameliorate or abrogate high-dose chemotherapy-induced thrombocytopenia. Therefore, the ex vivo expansion of CD34(+) PBPC was systematically studied aiming for an optimum production of megakaryocytic cells. MATERIALS AND METHODS CD34(+) PBPC were cultured in serum-free medium comparing different (n = 23) combinations of stem cell factor (SCF) (S), IL-1beta (1), IL-3 (3), IL-6 (6), erythropoietin (EPO) (E), thrombopoietin (TPO) (T) and promegapoietin (PMP, a novel chimeric IL-3/TPO receptor agonist). Ex vivo-generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. RESULTS Addition of TPO to cultures stimulated with S163E, a potent progenitor cell expansion cocktail previously described by our group, effectively induced the generation of CD61(+) cells (day 12: 31.4 +/- 7.9%). The addition of PMP tended to be more effective than TPO +/- IL-3. Whereas EPO was not required to maximize TPO- or PMP-induced megakaryocytic cell production, the use of IL-6 and IL-1beta augmented cellular expansion as well as CD61(+) cell production rates in the majority of cytokine combinations studied. Thus, the most effective CD61(+) cell expansion cocktail consisted of S163 + PMP which resulted in 65.9 +/- 3.0% CD61(+) at day 12 and an overall production of 40.7 +/- 4.5 CD61(+) cells per seeded CD34(+) PBPC. However, the basic 2-factor combination S + PMP also allowed for an effective CD61(+) cell production (day 12 CD61(+) cell production: 15.1 +/- 1.6). Moreover, maximum amplification of CFU-Meg was observed after 7 days using this two-factor cocktail (12.9 +/- 2.6-fold). The majority of CD61(+) cells generated in TPO- or PMP-based medium were low-ploidy 4N and 8N cells, and ex vivo-generated CD61(+), CD41(+), and CD42b(+) cells were mainly double positive for FACS-measured intracellular von Willebrand Factor (vWF) (76.7 +/- 3.3%, 58.8 +/- 4.4%, and 82.7 +/- 2.5%, respectively). CONCLUSIONS Taken together, this study demonstrates that megakaryocytic cells can be effectively produced ex vivo with as little as two-factors (SCF + PMP), an approach that might be favorably employed in a clinical expansion trial aiming to ameliorate high-dose chemotherapy-induced thrombocytopenia.

Characterization of Purified and Ex Vivo Manipulated Human Hematopoietic Progenitor and Stem Cells in Xenograft Recipients

Abstract: Research on the biology, regulation, and transplantation of human hematopoietic stem cells requires test systems for the detection, monitoring, and quantitation of these cells. Xenografted animal models provide suitable stem cell assays, since they allow long‐term engraftment, multilineage differentiation, and serial transfer of human hematopoietic cells. Recent techniques for the separation of hematopoietic cells have provided highly purified cellular subsets selected on the basis of the surface marker phenotype. The stem cell content of these subsets, however, is still unclear. Also, innovative approaches for the induction of hematopoietic cell proliferation and differentiation have generated ex vivo manipulated cells whose biological properties and functions still remain to be assessed. This paper reports on the biological characterization of these cell populations by the use of xenograft models.

Ex Vivo Manipulation of Peripheral Blood CD34 + Cells

The success of autologous peripheral blood progenitor cell (PBPC) transplantation is challenged by relapse of malignant disease which might — at least in part — be mediated by graft contaminating tumor cells. Although the clinical role of tumor cell depletion still remains to be demonstrated in prospective, randomized trials, multiple purging strategies are currently pursued in the context of autologous stem cell transplantation. This report is discussing ex vivo manipulations of PBPC transplants with respect to purging of tumor cells, including the positive selection of CD34+ cells with or without negative depletion as well as ex vivo expansion techniques. In addition, adoptive immunotherapy strategies using ex vivo generated autologous dendritic cells for the treatment of minimal residual disease after stem cell transplantation will be discussed.