GPR105 message was detected in antibody staining cDNA was in vitro transcribed and translated

GPR105 message was detected in antibody staining cDNA was in vitro transcribed and translated. CD34+ CD38C bracket represent the round of subtraction; the lanes in the CD34+ CD38+ bracket represent independent samples. (coincides with the GPR receptor cluster on Chromosome 3 (Napolitano et al. PF-04937319 1996). Isolation and sequencing of the murine cDNA demonstrated PF-04937319 high cross-species homology, with a human:murine similarity of 90% and an identity of 81% at the amino acid level (Fig. 1C). Northern blots of human tissues demonstrated abundant signal in the heart, placenta, and smooth muscle, with minimal detectable signal in spleen, lymph node, and thymus (Fig. 2A). Of note, two bands were seen in placenta, although the basis for this is unknown. Further characterization of expression within human hematopoietic cells used immunophenotypic populations sorted to high purity by FACSor, in the case of G0 cells, by the selected suicide strategy of CD34+ cells noted above. Probing cDNA from subpopulations of bone-marrow-derived stem or progenitor PF-04937319 populations or fully mature blood cells demonstrated that the expression of GPR105 is highly restricted to the suicide-selected G0 CD34+ CD38C bone marrow cells (Fig. 2B). Open in a separate window Figure 2. GPR105 is restricted in tissue expression and within hematopoietic cells is limited to primitive cells. (cDNA and hybridization determined by autoradiography. Size markers are indicated in kilobases. (mRNA by semiquantitative RTCPCR. GPR105 message was detected in antibody staining cDNA was in vitro transcribed and translated. (mRNA as demonstrated by semiquantitative RTCPCR. Cells PF-04937319 isolated by cell sorting were assessed using 3 103 cells of each type and analyzed for or mRNA; control refers to a no-cDNA template. (= 6; = 0.00003). (panel). The fractional proportion of week 2 colonies is provided in the accompanying table. Cells were plated at 3C6 twofold dilutions in replicate wells and scored each week. Data shown are one representative of three independent experiments with comparable results. We next assessed the functional phenotype of GPR105+ versus GPR105C CD34+ CD38C subpopulations PF-04937319 isolated by cell sorting from human fetal bone marrow and assayed for lineage-committed progenitor cell function by measuring colony-forming cells (CFCs) in methycellulose. Stem cell function was measured in parallel by long-term culture cobblestone area formation (CAFC) or long-term culture-initiating cell (LTC-IC) assays on bone marrow stroma. Among CD34+ CD38C cells, GPR105+ cells performed poorly compared with GPR105C cells in the CFC assays (mean 4.2 vs. 42.0 in 6 independent experiments, = 0.00003; Fig. 4B). Poor colony production could indicate either that GPR105+ cells are simply unable to grow in methylcellulose, are postmitotic, terminally differentiated cells, or are a more primitive, relatively cytokine-unresponsive stem cell subset. Long-term assays performed using limit dilutions of cells cultured on primary human marrow stroma discriminated between the latter two. Three independent experiments scored weekly demonstrated low cobblestone area production with the human GPR105+ cells (Fig. 4C). However, human GPR105+ cells demonstrated a marked difference in capacity to provide a sustained output of hematopoietic colonies (= 0.008). In contrast to human GPR105C cells, with which CAFC production declined over time as would be expected from a predominantly progenitor population (91% decline by week 8), GPR105+ cells consistently demonstrated sustained cobblestone formation over the same interval (47% decline; Fig. 4C). When the CAFC cultures were overlaid with cytokine-supplemented methlycellulose at week 5 (LTC-IC assay), colonies emerged, which were micropippetted and evaluated morphologically. Cells with myeloid cell histologic appearance were observed (Fig. 4D). Thus, low-level mature cell production was ongoing for prolonged intervals, a characteristic consistent with a stem cell phenotype. Although KLRC1 antibody ectopic expression experiments need to be interpreted with caution, when we transduced primary human CD34+ cells with either a GPR105-encoding or a control retroviral vector with a bicistronic GFP, we noted a shift in the functional phenotype. Progenitor function as measured by CFC declined (= 0.02; Fig. 4E), whereas stem cell function as measured by LTC-IC or CAFC increased (= 0.03; Fig. 4F). This was.

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