Michael Punzel

The symmetry of initial divisions of human hematopoietic progenitors is altered only by the cellular microenvironment

OBJECTIVE We examined if cellular elements or adhesive ligands were able to alter asymmetric divisions of CD34(+)/CD38(-) cells in contrast to soluble factors at a single cell level. MATERIALS AND METHODS After single cell deposition onto 96-well plates, cells were cocultured for 10 days with the stem cell supporting cell line AFT024, fibronectin (FN), or bovine serum albumin (BSA). The divisional history was monitored with time-lapse microscopy. Subsequent function for the most primitive cells was assessed using the myeloid-lymphoid-initiating cell (ML-IC) assay. Committed progenitors were measured using colony-forming cells (CFC). RESULTS Only contact with AFT024 recruited significant numbers of CD34(+)/CD38(-) cells into cell cycle and increased asymmetric divisions. Although most ML-IC were still identified among cells that have divided fewer than 3 times, a significant number of ML-IC shifted into the fast-dividing fraction after exposure to AFT024. The increase in ML-IC frequency was predominantly due to recruitment of quiescent and slow-dividing cells from the starting population. Increase in CFC activity induced by AFT024 was found only among rapidly dividing cells. CONCLUSIONS For the first time, we have demonstrated that asymmetric divisions can be altered upon exposure with a stem cell-supporting microenvironment. For the primitive subset of cells (ML-IC), this was predominantly due to recruitment into cell cycle and increased rounds of cycling without loss of function. Exposure to AFT024 cells also increased proliferation and asymmetric divisions of committed CFC. Hence direct communication between hematopoietic progenitors with stroma cells is required for maintaining self-renewal potential.

Functional analysis of initial cell divisions defines the subsequent fate of individual human CD34+CD38− cells

OBJECTIVE We assessed the relationship of individual cell divisional behavior with the functional fate of stem cell candidates at the single cell level. MATERIALS AND METHODS Individual CD34(+)CD38(-) cells derived from cord blood (88-352 cells in each of 25 experiments) were cultured in early-acting conditioned medium (EACM) or late-acting proliferation medium (LAPM). The initial cell divisions were microscopically monitored every 12 to 24 hours and then assessed for primitive function in the myeloid lymphoid-initiating cell assay and committed function in the colony-forming cell (CFC) assay. RESULTS Despite a higher proliferative capacity in LAPM, significantly more quiescent cells (11.1 +/- 1.7%) were found in LAPM than in EACM cultures (1.1 +/- 0.4%; p < 0.001). No differences were observed in the initially plated CD34(+)Cd38(-) cells that produced asymmetrically dividing progeny. The majority of cells with subsequent ML-IC function divided in EACM but were found among quiescent cells in LAPM conditions. All cycling cells with subsequent ML-IC capacity initially remained quiescent for at least 96 hours. All ML-IC had been recruited exclusively (100%) from either cytokine nonresponsive (quiescent) or slow and asymmetrically dividing cells (1-2 divisions). In contrast, the majority of CFCs entered the cell cycle immediately after plating, have divided more than two times, and only 20.2 +/- 5.5% of the cycled CFC divided asymmetrically. CONCLUSIONS Asymmetric divisional behavior of CD34(+)CD38(-)cells cannot be influenced by culture conditions. Primitive ML-IC can be distinguished from committed CFC by initial quiescence or asymmetric divisions. Committed CFC cycle rapidly and symmetrically.

Hematopoietic stem cells: can old cells learn new tricks?

Since the establishment of cell lines derived from human embryonic stem (ES) cells, it has been speculated that out of such “raw material,” we could some day produce all sorts of replacement parts for the human body. Human pluripotent stem cells can be isolated from embryonic, fetal, or adult tissues. Enormous self‐renewal capacity and developmental potential are the characteristics of ES cells. Somatic stem cells, especially those derived from hematopoietic tissues, have also been reported to exhibit developmental potential heretofore not considered possible. The initial evidences for the plasticity potential of somatic stem cells were so encouraging that the opponents of ES cell research used them as arguments for restricting ES cell research. In the past months, however, critical issues have been raised challenging the validity and the interpretation of the initial data. Whereas hematopoietic stem‐cell therapy has been a clinical reality for almost 40 years, there is still a long way to go in basic research before novel therapy strategies with stem cells as replacement for other organ systems can be established. Given the present status, we should keep all options open for research in ES cells and adult stem cells to appreciate the complexity of their differentiation pathways and the relative merits of various types of stem cells for regenerative medicine.

Divisional History and Pluripotency of Human Hematopoietic Stem Cells

Abstract: To maintain self‐renewal and multilineage differentiation capacity, hematopoietic stem cell (HSC) proliferation requires both symmetric and asymmetric cell divisions. We have applied a time‐lapse camera system and our single‐cell culture to correlate early replication behavior with short‐ and long‐term function. Using five‐dimensional flow cytometry to purify subpopulations of fetal liver (FLV), fetal bone marrow (FBM), umbilical cord blood (UCB), adult bone marrow (ABM), and mobilized peripheral blood (MPB), we studied the relationship between colony efficiency (CE) growth pattern and ontogenic age. The highest CE was found among HSC candidates from FLV, FBM, and UCB and the lowest from ABM. Relating the divisional behavior with functional readouts, we demonstrated that although mitotic rate, colony efficiency, and percent of asymmetric divisions all decreased with ontogenic age, the fraction of cells undergoing asymmetric divisions was consistently at 45%. After 10 days of culture, 60.6 ± 9.8% of the PKH bright cells gave rise to colonies (15.8 ± 7.8% dispersed) compared to 15.9 ± 11.1% of the PKH dim cells (2.5 ± 2.5% dispersed). In addition, the much more primitive Myeloid‐Lymphoid Initiating Cells (ML‐IC) are predominantly found in the PKH‐bright population. Thus, primitive function of individual candidate HSCs closely related to their divisional behavior.

Lineage development of hematopoietic stem and progenitor cells

Abstract Hematopoietic stem cells have the potential to develop into multipotent and different lineage-restricted progenitor cells that subsequently generate all mature blood cell types. The classical model of hematopoietic lineage commitment proposes a first restriction point at which all multipotent hematopoietic progenitor cells become committed either to the lymphoid or to the myeloid development, respectively. Recently, this model has been challenged by the identification of murine as well as human hematopoietic progenitor cells with lymphoid differentiation capabilities that give rise to a restricted subset of the myeloid lineages. As the classical model does not include cells with such capacities, these findings suggest the existence of alternative developmental pathways that demand the existence of additional branches in the classical hematopoietic tree. Together with some phenotypic criteria that characterize different subsets of multipotent and lineage-restricted progenitor cells, we summarize these recent findings here.

Cytokine combinations differentially influence the SDF-1α-dependent migratory activity of cultivated murine hematopoietic stem and progenitor cells

Abstract Stromal cell-derived factor-1α (SDF-1α) is a strong migratory stimulant for hematopoietic stem and progenitor cells (HSPCs). The hematopoietic cytokines thrombopoietin (TPO), Flt3-ligand (FL), stem cell factor (SCF) and interleukin 11 (IL-11) are able to stimulate amplification of primitive murine hematopoietic stem cells (HSCs) in vitro. The effects of these cytokines on SDF-1α-induced migratory activity of murine Lin-c-kit+ HSPC were analyzed by cultivation of these cells in the presence of 12 combinations of FL, TPO, SCF and IL-11. Migratory activity was measured in a three-dimensional collagen matrix using time-lapse video microscopy. Each cytokine combination had a distinct effect on SDF-1α-stimulated migratory activity. For instance, FL- and SCF-cultivated cells showed a high migratory SDF-1α response, while cells cultivated with SCF, TPO and IL-11 did not react to SDF-1α stimulation with an elevated migration rate. Our data indicate that the differences in the migratory SDF-1α response are not related to different CXCR4 expression levels, but rather to the differential engagement of the CXCR4-dependent MAPKp42/44 and PI3K signal transduction pathways. This indicates that hematopoietic cytokines can have a significant impact on SDF-1α-stimulated migratory activity and the underlying intracellular signaling processes in cultivated HSPCs.

Dengue virus transmission by blood stem cell donor after travel to Sri Lanka; Germany, 2013.

Three days after donation of peripheral blood stem cells to a recipient with acute myeloblastic leukemia, dengue virus was detected in the donor, who had recently traveled to Sri Lanka. Transmission to the recipient, who died 9 days after transplant, was confirmed.

Detection of HBV DNA in the blood of a stem cell donor after G‐CSF treatment

Occult hepatitis B virus (HBV) infections have gained much attention in terms of transmissibility and reactivation. Here, we report the case of a healthy stem cell donor with serological evidence of a resolved HBV infection who became temporarily HBV DNA–positive in the blood after stimulation with granulocyte colony-stimulating factor (G-CSF). A 51-year-old man from the German Bone Marrow Donor Center was identified as human leukocyte antigen matching (10/10) stem cell donor for a 44-year-old female patient diagnosed with precursor B-cell acute lymphoblastic leukemia in the United Kingdom. This donor was a well-known blood donor since 2009 and was previously tested positive for antibody to hepatitis B core antigen, antibody to hepatitis B e antigen, and antibody to hepatitis B surface antigen (anti-HBs). During the diagnostic workup the serological findings could be confirmed and HBV DNA was not detected. Thus, in accordance with German regulations, this donor was considered eligible for stem cell donation. The donor started G-CSF treatment, and at day 5 cluster of differentiation 34 (CD34)–positive stem and progenitor cells were collected. At this time HBV DNA (pooled tested, detection limit 257 IU/mL) tested negative. The product was shipped from the German apheresis center to the UK transplantation center and infused after myeloablative conditioning of the patient had been completed. Six days after transplantation, testing of the product at the transplantation center revealed the presence of 26 IU/mL HBV DNA, which could be confirmed by testing backup samples (product and blood) (Table 1; Supporting Information). Therefore, the stem cell recipient immediately started lamivudine treatment and has tested negative for HBV DNA (last test day 1130) ever since. AntiHBs of the recipient was negative before transplantation but temporarily positive after transplantation (Supporting Information). HBV DNA testing of the donor was again negative at day 16 after apheresis. Anti-HBs titration and neutralizing experiments using hepatitis B surface antigen (HBsAg) from different genotypes specified the presence 3,300 IU/L anti-HBs in the blood of the donor highly capable of neutralizing HBsAg from different genotypes. Parts of the HBV DNA detected in the