Surfaceome profiling reveals regulators of neural stem cell function.

The composition of cell-surface proteins changes during lineage specification, altering cellular responses to their milieu. The changes that characterize maturation of early neural stem cells (NSCs) remain poorly understood. Here we use mass spectrometry-based cell surface capture technology to profile the cell surface of early NSCs and demonstrate functional requirements for several enriched molecules. Primitive NSCs arise from embryonic stem cells upon removal of Transforming growth factor-ß signaling, while definitive NSCs arise from primitive NSCs upon Lif removal and FGF addition. In vivo aggregation assays revealed that N-cadherin upregulation is sufficient for the initial exclusion of definitive NSCs from pluripotent ectoderm, while c-kit signaling limits progeny of primitive NSCs. Furthermore, we implicate EphA4 in primitive NSC survival signaling and Erbb2 as being required for NSC proliferation. This work elucidates several key mediators of NSC function whose relevance is confirmed on forebrain-derived populations and identifies a host of other candidates that may regulate NSCs.

Measurement of generation-dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation.

Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G(0) -G(5) ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G(2) to G(5) and higher death rates in G(2) , G(3) , and G(5) compared with SCF. In addition, proliferation rates tended to increase from G(1) to G(5) in cultures supplemented with EPO and EPO + SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture.

Biochemical measurements on single erythroid progenitor cells shed light on the combinatorial regulation of red blood cell production.

Adult bone marrow (BM) erythrocyte colony-forming units (CFU-Es) are important cellular targets for the treatment of anemia and also for the manufacture of red blood cells (RBCs) ex vivo. We obtained quantitative biochemical measurements from single and small numbers of CFU-Es by isolating and analyzing c-Kit(+)CD71(high)Ter119(-) cells from adult mouse BM and this allowed us to identify two mechanisms that can be manipulated to increase RBC production. As expected, maximum RBC output was obtained when CFU-Es were stimulated with a combination of Stem Cell Factor (SCF) and Erythropoietin (EPO) mainly because SCF supports a transient CFU-E expansion and EPO promotes the survival and terminal differentiation of erythroid progenitors. However, we found that one of the main factors limiting the output in RBCs was that EPO induces a downregulation of c-Kit expression which limits the transient expansion of CFU-Es. In the presence of SCF, the EPO-mediated downregulation of c-Kit on CFU-Es is delayed but still significant. Moreover, treatment of CFU-Es with 1-Naphthyl PP1 could partially inhibit the downregulation of c-Kit induced by EPO, suggesting that this process is dependent on a Src family kinase, v-Src and/or c-Fyn. We also found that CFU-E survival and proliferation was dependent on the level of time-integrated extracellular-regulated kinase (ERK) activation in these cells, all of which could be significantly increased when SCF and EPO were combined with mouse fetal liver-derived factors. Taken together, these results suggest two novel molecular strategies to increase RBC production and regeneration.