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• W2143869444 abstract "In hematopoiesis, co-expression of Sca-1 and c-Kit defines cells (LS+K) with long term reconstituting potential. In contrast, poorly characterized LS−K cells fail to reconstitute lethally irradiated recipients. Relative quantification mass spectrometry and transcriptional profiling were used to characterize LS+K and LS−K cells. This approach yielded data on >1200 proteins. Only 32% of protein changes correlated to mRNA modulation demonstrating post-translational protein regulation in early hematopoietic development. LS+K cells had lower expression of protein synthesis proteins but did express proteins associated with mature cell function. Major increases in erythroid development proteins were observed in LS−K cells; based on this assessment of erythroid potential we showed them to be principally erythroid progenitors, demonstrating effective use of discovery proteomics for definition of primitive cells. In hematopoiesis, co-expression of Sca-1 and c-Kit defines cells (LS+K) with long term reconstituting potential. In contrast, poorly characterized LS−K cells fail to reconstitute lethally irradiated recipients. Relative quantification mass spectrometry and transcriptional profiling were used to characterize LS+K and LS−K cells. This approach yielded data on >1200 proteins. Only 32% of protein changes correlated to mRNA modulation demonstrating post-translational protein regulation in early hematopoietic development. LS+K cells had lower expression of protein synthesis proteins but did express proteins associated with mature cell function. Major increases in erythroid development proteins were observed in LS−K cells; based on this assessment of erythroid potential we showed them to be principally erythroid progenitors, demonstrating effective use of discovery proteomics for definition of primitive cells. Hematopoiesis generates mature cells of many different lineages via a process of differentiation and development from a common multipotent hematopoietic stem cell (HSC). 1The abbreviations used are: HSC, hematopoietic stem cell; LS+K, Lin−Sca-1+Kit+; LS−K, Lin−Sca-1−Kit+; iTRAQ, isobaric tagging for relative and absolute quantitation; HPP-CFC, high proliferative potential colony-forming cell; LPP-CFC, low proliferative potential colony-forming cells; CFU-e, colony-forming unit-erythroid; hnRNP, heterogenous nuclear ribonucleoprotein. Although the mechanisms that govern these processes remain to be fully elucidated, two experimental approaches, namely the establishment of rigorous clonal assays to measure HSC function in animal models and the development of methodologies for the prospective isolation of these rare cells, have contributed to HSC being among the best understood population of tissue stem cells in adult vertebrate physiology. Over the past 25 years, a wide variety of strategies have been developed for the prospective isolation of HSC. Such is the state of the art of HSC purification that several groups have reported reconstitution of mice with a single prospectively isolated HSC (1Christensen J.L. Weissman I.L. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14541-14546Crossref PubMed Scopus (617) Google Scholar, 2Goodell M.A. Brose K. Paradis G. Conner A.S. Mulligan R.C. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.J. Exp. Med. 1996; 183: 1797-1806Crossref PubMed Scopus (2495) Google Scholar, 3Okada S. Nakauchi H. Nagayoshi K. Nishikawa S.I. Miura Y. Suda T. In vivo and in vitro stem-cell function of c-Kit-positive and Sca-1-positive murine hematopoietic-cells.Blood. 1992; 80: 3044-3050Crossref PubMed Google Scholar, 4Osawa M. Hanada K. Hamada H. Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34− low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1729) Google Scholar, 5Osawa M. Nakamura K. Nishi N. Takahashi N. Tokuomoto Y. Inoue H. Nakauchi H. In vivo self-renewal of c-Kit(+) Sca-1(+) Lin(low/−) hemopoietic stem cells.J. Immunol. 1996; 156: 3207-3214PubMed Google Scholar, 6Randall T.D. Lund F.E. Howard M.C. Weissman I.L. Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.Blood. 1996; 87: 4057-4067Crossref PubMed Google Scholar, 7Spangrude G.J. Heimfeld S. Weissman I.L. Purification and characterization of mouse hematopoietic stem-cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2235) Google Scholar). The expression of a range of cell surface markers has been shown to enable enrichment of cells with long term reconstituting capacity (1Christensen J.L. Weissman I.L. Flk-2 is a marker in hematopoietic stem cell differentiation: a simple method to isolate long-term stem cells.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14541-14546Crossref PubMed Scopus (617) Google Scholar, 2Goodell M.A. Brose K. Paradis G. Conner A.S. Mulligan R.C. Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.J. Exp. Med. 1996; 183: 1797-1806Crossref PubMed Scopus (2495) Google Scholar, 3Okada S. Nakauchi H. Nagayoshi K. Nishikawa S.I. Miura Y. Suda T. In vivo and in vitro stem-cell function of c-Kit-positive and Sca-1-positive murine hematopoietic-cells.Blood. 1992; 80: 3044-3050Crossref PubMed Google Scholar, 4Osawa M. Hanada K. Hamada H. Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34− low/negative hematopoietic stem cell.Science. 1996; 273: 242-245Crossref PubMed Scopus (1729) Google Scholar, 5Osawa M. Nakamura K. Nishi N. Takahashi N. Tokuomoto Y. Inoue H. Nakauchi H. In vivo self-renewal of c-Kit(+) Sca-1(+) Lin(low/−) hemopoietic stem cells.J. Immunol. 1996; 156: 3207-3214PubMed Google Scholar, 6Randall T.D. Lund F.E. Howard M.C. Weissman I.L. Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.Blood. 1996; 87: 4057-4067Crossref PubMed Google Scholar, 7Spangrude G.J. Heimfeld S. Weissman I.L. Purification and characterization of mouse hematopoietic stem-cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2235) Google Scholar). In the mouse, the most commonly utilized methodology for isolation of HSC was developed by Weissman and co-workers (7Spangrude G.J. Heimfeld S. Weissman I.L. Purification and characterization of mouse hematopoietic stem-cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2235) Google Scholar). According to this strategy, HSCs are restricted to a subpopulation of bone marrow cells that lack expression of mature hematopoietic cell lineage markers such as Mac1 (macrophage marker), Gr-1 (granulocytic marker), CD4/CD8 and B220 (T and B lymphoid markers, respectively), and Ter119 (erythroid marker) (6Randall T.D. Lund F.E. Howard M.C. Weissman I.L. Expression of murine CD38 defines a population of long-term reconstituting hematopoietic stem cells.Blood. 1996; 87: 4057-4067Crossref PubMed Google Scholar, 8Bertoncello I. Bradley T.R. Watt S.M. An improved negative immunomagnetic selection strategy for the purification of primitive hematopoietic-cells from normal bone-marrow.Exp. Hematol. 1991; 19: 95-100PubMed Google Scholar). Within this lineage-negative (Lin−) fraction, primitive hematopoietic stem and progenitor cells are further identified by their expression of Sca-1 (Ly-6E) and the receptor tyrosine kinase c-Kit (stem cell factor receptor). c-Kit is present on both primitive hematopoietic cells and their immediate progeny (3Okada S. Nakauchi H. Nagayoshi K. Nishikawa S.I. Miura Y. Suda T. In vivo and in vitro stem-cell function of c-Kit-positive and Sca-1-positive murine hematopoietic-cells.Blood. 1992; 80: 3044-3050Crossref PubMed Google Scholar). c-Kit mutant mice are sterile, lack melanocytes, and have defects in their hematopoiesis associated with a macrocytic anemia (9Huang E. Nocka K. Beier D.R. Chu T.Y. Buck J. Lahm H.W. Wellner D. Leder P. Besmer P. The hematopoietic growth factor-KL is encoded by the Si-locus and is the ligand of the c-Kit receptor, the gene-product of the W-locus.Cell. 1990; 63: 225-233Abstract Full Text PDF PubMed Scopus (941) Google Scholar, 10Zsebo K.M. Williams D.A. Geissler E.N. Broudy V.C. Martin F.H. Atkins H.L. Hsu R.Y. Birkett N.C. Okino K.H. Murdock D.C. Jacobsen F.W. Langley K.E. Smith K.A. Takeishi T. Cattanach B.M. Galli S.J. Suggs S.V. Stem-cell factor is encoded at the Si-Locus of the mouse and is the ligand for the c-Kit tyrosine kinase receptor.Cell. 1990; 63: 213-224Abstract Full Text PDF PubMed Scopus (1216) Google Scholar). Sca-1 is a glycosylphosphatidylinositol-linked cell surface protein with a potential role in membrane protein organization for signaling via lipid raft structures (11Horejsi V. Drbal K. Cebecauer M. Cerny J. Brdicka T. Angelisova P. Stockinger H. GPI-microdomains: a role in signalling via immunoreceptors.Immunol. Today. 1999; 20: 356-361Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). It has also been implicated in Src protein-tyrosine kinase family signaling and in the regulation of integrin function in stem cells (12Epting C.L. Lopez J.E. Shen X. Liu L.S. Bristow J. Bernstein H.S. Stem cell antigen-1 is necessary for cell-cycle withdrawal and myoblast differentiation in C2C12 cells.J. Cell Sci. 2004; 117: 6185-6195Crossref PubMed Scopus (51) Google Scholar, 13Bradfute S.B. Graubert T.A. Goodell M.A. Roles of Sca-1 in hematopoietic stem/progenitor cell function.Exp. Hematol. 2005; 33: 836-843Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Stefanova I. Horejsi V. Ansotegui I.J. Knapp W. Stockinger H. GPI-anchored cell-surface molecules complexed to protein tyrosine kinases.Science. 1991; 254: 1016-1019Crossref PubMed Scopus (769) Google Scholar). Sca-1 expression is a marker enabling enrichment of stem cells (3Okada S. Nakauchi H. Nagayoshi K. Nishikawa S.I. Miura Y. Suda T. In vivo and in vitro stem-cell function of c-Kit-positive and Sca-1-positive murine hematopoietic-cells.Blood. 1992; 80: 3044-3050Crossref PubMed Google Scholar, 7Spangrude G.J. Heimfeld S. Weissman I.L. Purification and characterization of mouse hematopoietic stem-cells.Science. 1988; 241: 58-62Crossref PubMed Scopus (2235) Google Scholar) as demonstrated by the capacity of Lin−Sca-1+Kit+ (LS+K) cells to reconstitute multilineage hematopoiesis in lethally irradiated mice, whereas the corresponding Lin−Sca-1−Kit+ (LS−K) population fail to do so (3Okada S. Nakauchi H. Nagayoshi K. Nishikawa S.I. Miura Y. Suda T. In vivo and in vitro stem-cell function of c-Kit-positive and Sca-1-positive murine hematopoietic-cells.Blood. 1992; 80: 3044-3050Crossref PubMed Google Scholar). In Sca-1-null murine bone marrow there was a decreased number of hematopoietic progenitor cells and a reduced ability of Sca-1-null cells to repopulate hematopoiesis in lethally irradiated mice. HSC from Sca-1-null mice with a Kit mutation at a single allele display a profound anemia with reduced progenitor cell numbers (15Ito C.Y. Li C.Y.J. Bernstein A. Dick J.E. Stanford W.L. Hematopoietic stem cell and progenitor defects in Sca-1/Ly-6A-null mice.Blood. 2003; 101: 517-523Crossref PubMed Scopus (150) Google Scholar), far greater than observed in Sca-1-null animals, thus demonstrating the critical and linked role for both these proteins in hematopoiesis. Indeed there is evidence that Sca-1 expression can modulate the expression of c-Kit (13Bradfute S.B. Graubert T.A. Goodell M.A. Roles of Sca-1 in hematopoietic stem/progenitor cell function.Exp. Hematol. 2005; 33: 836-843Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Although the nature of the LS+K cells is defined by their ability to promote long term reconstitution in lethally irradiated recipient mice, the LS−K cells have not been characterized beyond their lack of ability to rescue myeloablated mice. Subsequent studies have defined a discrete subpopulation of cells with progenitor cell activity within the LS−K compartment such as common myeloid progenitor cells (IL-7Rα−, FcγRlow, CD34+, Lin−, Sca−, Kit+ cells) (16Akashi K. Traver D. Miyamoto T. Weissman I.L. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.Nature. 2000; 404: 193-197Crossref PubMed Scopus (1913) Google Scholar). However, the characteristics of the majority of the cells await definition. We have shown previously that systematic proteomics analysis of primary hematopoietic stem cells is feasible on small populations of cells (∼1 million) using an isobaric tagging procedure for relative quantification coupled to two-dimensional LC plus MS/MS (17Unwin R.D. Pierce A. Watson R.B. Sternberg D.W. Whetton A.D. Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.Mol. Cell. Proteomics. 2005; 4: 924-935Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). This approach revealed that post-translational regulation of protein expression is critically important in hematopoietic cell development, and therefore many transcriptomic changes observed do not affect the proteome (17Unwin R.D. Pierce A. Watson R.B. Sternberg D.W. Whetton A.D. Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.Mol. Cell. Proteomics. 2005; 4: 924-935Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 18Unwin R.D. Smith D.L. Blinco D. Wilson C.L. Miller C.J. Evans C.A. Jaworska E. Baldwin S.A. Barnes K. Pierce A. Spooncer E. Whetton A.D. Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells.Blood. 2006; 107: 4687-4694Crossref PubMed Scopus (151) Google Scholar). Similarly alterations in the proteome occur in the absence of significant changes in transcript expression. To define the Lin−Sca−Kit+ population we compared its proteome with that of Lin−Sca+Kit+ cells and demonstrate that systematic analysis of enriched hematopoietic populations can give a direct insight into their biological characteristics. C57Bl/6J mice were purchased from Animal Resources Centre (Perth, Western Australia, Australia) and housed for at least a week prior to experimental use. Murine bone marrow was collected from femora, tibiae, and iliac crests. Lineage marker-depleted cells were enriched as described previously (19Nilsson S.K. Johnston H.M. Coverdale J.A. Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches.Blood. 2001; 97: 2293-2299Crossref PubMed Scopus (473) Google Scholar). Briefly low density (<1.077 g/cm3) cells were labeled with a mixture of biotinylated rat anti-mouse antibodies: B220, macrophage antigen-1 (Mac-1), Gr-1, CD4, CD8, CD3, CD5, and Ter119. Lineage marker-positive cells were removed by immunomagnetic selection using magnetic activated cell separation (Miltenyi Biotec, Bergisch, Gladbach, Germany). Lineage marker-negative cells were labeled with Sca-1-FITC (1 μg/5 × 106 cells; Pharmingen), c-Kit-phycoerythrin (1 μg/5 × 106 cells; Pharmingen), and streptavidin-Red 670 (1:160 final concentration; Invitrogen). LS−K and LS+K cells were isolated by flow cytometry as described previously (19Nilsson S.K. Johnston H.M. Coverdale J.A. Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches.Blood. 2001; 97: 2293-2299Crossref PubMed Scopus (473) Google Scholar) and stored as dry pellets at −80 °C prior to mass spectrometric analysis. Methods used for hematopoietic cell analyses were as described previously (18Unwin R.D. Smith D.L. Blinco D. Wilson C.L. Miller C.J. Evans C.A. Jaworska E. Baldwin S.A. Barnes K. Pierce A. Spooncer E. Whetton A.D. Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells.Blood. 2006; 107: 4687-4694Crossref PubMed Scopus (151) Google Scholar), and the experiment was run in triplicate. Briefly cells were solubilized in 0.5 m triethylammonium bicarbonate (Sigma) + 0.05% (w/v) SDS on ice for 20 min. Protein (90 μg in 100 μl) was reduced by addition of 5 μl of 50 mm tris(2-carboxyethyl)phosphine, and reduced cysteine residues were then blocked by addition of 2.5 μl of 200 mm methylmethanethiosulfate in isopropanol. Protein was digested by addition of 18 μl of trypsin at 0.5 μg/μl and incubated at 37 °C overnight. Peptides were dried, reconstituted in 20 μl 0.5 m triethylammonium bicarbonate, and labeled with iTRAQ reagent (Applied Biosystems Inc., Framingham, MA) as described previously (17Unwin R.D. Pierce A. Watson R.B. Sternberg D.W. Whetton A.D. Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.Mol. Cell. Proteomics. 2005; 4: 924-935Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). For separation prior to reversed phase LC-MS/MS peptides were fractionated into ∼50 fractions off line using an strong cation exchange column (10 × 2.1-cm PolyLC Polysulfoethyl A column, 5-μm beads, 200-Å pore size; Hichrom Ltd., Reading, Berks, UK) on a Dionex LC system using the gradient described previously (18Unwin R.D. Smith D.L. Blinco D. Wilson C.L. Miller C.J. Evans C.A. Jaworska E. Baldwin S.A. Barnes K. Pierce A. Spooncer E. Whetton A.D. Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells.Blood. 2006; 107: 4687-4694Crossref PubMed Scopus (151) Google Scholar). Dried peptide fractions were resuspended in 180 μl of 2% (v/v) acetonitrile, 0.1% (v/v) formic acid, and a 60-μl aliquot was loaded onto a 75-μm-inner diameter × 15-cm column packed with C18 PepMap100 (3 μm, 100 Å) using an LC Packings (Amsterdam, Netherlands) UltiMate™ pump and separated as described previously (17Unwin R.D. Pierce A. Watson R.B. Sternberg D.W. Whetton A.D. Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.Mol. Cell. Proteomics. 2005; 4: 924-935Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Data were acquired using an independent data acquisition protocol where an MS scan was taken and then the two highest ions were selected for fragmentation followed by dynamic exclusion for 1 min. Data were processed using ProQUANT software version 1.1 (Applied Biosystems) as described previously (18Unwin R.D. Smith D.L. Blinco D. Wilson C.L. Miller C.J. Evans C.A. Jaworska E. Baldwin S.A. Barnes K. Pierce A. Spooncer E. Whetton A.D. Quantitative proteomics reveals posttranslational control as a regulatory factor in primary hematopoietic stem cells.Blood. 2006; 107: 4687-4694Crossref PubMed Scopus (151) Google Scholar). Briefly data were searched against the murine International Protein Index (IPI) database version 3.13 (50,489 entries), allowing for iTRAQ labeling on lysines and N termini and cysteine modification with methylmethanethiosulfate with mass tolerances of 0.15 Da for MS and 0.1 Da for MS/MS, allowing for one missed (trypsin) cleavage, and including peptides with a minimum confidence score of 70. Quantitation was performed using the default software settings. ProGROUP (Applied Biosystems Inc.) software was used to assign a single accession number for each protein. These outputs are available as supplemental data and provide alternative names and accession numbers for proteins that match to the identified peptides. For a protein to be considered significantly different between LS+K and LS−K cells, at least three (non-unique) peptides had to be identified and quantified, the -fold change had to be >1.2, that protein had to have a p < 0.05 from a Student's t test in two of three analyses, and the LS−K1 versus LS−K2 and LS+K1 versus LS+K2 ratios had to be between 0.92 and 1.10 in all replicates where data were obtained for that protein. Total cellular RNA was prepared from 1 × 104 freshly isolated cells of each cell population using RNeasy minicolumns (Qiagen) according to the manufacturer's instructions. RNA was amplified and labeled using the Two-cycle Target Labeling and Control Reagents (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions. 15 μg of biotinylated amplified cRNA were hybridized onto Affymetrix GeneChip arrays (MOE430 Plus-2.0, Affymetrix). Arrays were scanned using Gene Array Scanner (Affymetrix). Affymetrix GCOS 1.2 was used for data acquisition and analysis. The comparative analysis of results obtained for the different cell populations was performed using the Silicon Genetics GeneSpring GX 7.3.1 software (Agilent Technologies, Palo Alto, CA). Affymetrix CEL files were normalized using the GC-RMA method. A transcript was determined to be “significantly differentially expressed” if it was called “present” in all three replicate arrays and had a -fold change of ±1.8 that was deemed to be different (p < 0.05) by Student's t test. To compare transcriptome and proteome data, IPI protein accession numbers were used to identify the relevant probe set identifiers from the microarray data set using the NetAffx tool (Affymetrix). Transplantation analysis was performed using C57Bl/6J female recipient mice and male donor LS+K or LS−K cells. Six weeks after engraftment, bone marrow was harvested, DNA was isolated, and the proportion of male donor was determined by using real time PCR targeting the Y6 amplicon on the Y chromosome as described previously (20Peters S.O. Bauermeister K. Simon J.P. Branke B. Wagner T. Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice.J. Immunol. Methods. 2002; 260: 109-116Crossref PubMed Scopus (23) Google Scholar) and modified by Nilsson et al. (21Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar). High proliferative potential colony-forming cells (HPP-CFCs) were assayed in a double layer nutrient agar culture system as described previously except that stem cell factor (100 ng/ml; Amgen, Thousand Oaks, CA) was added to CSF-1, IL-1, and IL-3 (21Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar, 22Bartelmez S.H. Bradley T.R. Bertoncello I. Mochizuki D.Y. Tushinski R.J. Stanley E.R. Hapel A.J. Young I.G. Kriegler A.B. Hodgson G.S. Interleukin-1 plus Interleukin-3 plus colony-stimulating factor-I are essential for clonal proliferation of primitive myeloid bone-marrow cells.Exp. Hematol. 1989; 17: 240-245PubMed Google Scholar). Two doses of 50 mg/kg busulfan were given subcutaneously 1 and 3 days prior to transplanting limiting numbers of LS+K and LS−K cells intravenously (10,000, 3000, 1000, and 300/mouse) (21Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar). Colony forming assays were performed essentially as described previously (21Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar). For committed erythroid and megakaryocytic colony formation, cells were plated at 1, 3, 10. and 30 cells/well in 100 μl of CellGro Good Manufacturing Practice serum-free medium (CellGenix, Antioch, IL) supplemented with 4 units/ml erythropoietin (Amgen). After 7 days the wells were scored for the presence of erythroid and megakaryocytic cells. A cluster of eight or more erythroid cells was defined as a colony-forming unit-erythroid (CFU-e). The incidence of progenitor cells was determined from the number of colonies formed using L-Calc software (StemSoft Software Inc.). The erythroid nature of cells in this assay was confirmed by flow cytometric analysis after Ter119 staining. Populations of LS+K and LS−K cells from murine bone marrow were isolated by means of flow cytometry according to the gates shown in Fig. 1A. LS+K cells constituted 0.017 ± 0.003% (mean ± S.E., n = 3) of the nucleated bone marrow cells, and LS−K cells constituted 0.44 ± 0.07% (mean ± S.E., n = 3). To confirm their long or short term repopulation potential, these populations were assessed for the ability to reconstitute busulfan-myeloablated animals (21Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar). After 6 weeks, no LS−K cell-derived cells were detected in the bone marrow of recipient animals transplanted with 30,000, 15,000, 3000, or 300 LS-K cells plus 300 LS+K cells. Similarly 30,000 LS−K cells transplanted in the absence of LS+K cells gave less than 0.5% donor cells in recipient animals after 6 weeks. As shown previously, transplantation of 300–10,000 LS+K cells was effective in re-establishing hematopoiesis and enabling recipient mice to survive busulfan myeloablation (21Nilsson S.K. Johnston H.M. Whitty G.A. Williams B. Webb R.J. Denhardt D.T. Bertoncello I. Bendall L.J. Simmons P.J. Haylock D.N. Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive hematopoietic progenitor cells.Blood. 2005; 106: 1232-1239Crossref PubMed Scopus (605) Google Scholar). The lack of the Sca-1 marker therefore indicated that this is a more mature cell population as expected (3Okada S. Nakauchi H. Nagayoshi K. Nishikawa S.I. Miura Y. Suda T. In vivo and in vitro stem-cell function of c-Kit-positive and Sca-1-positive murine hematopoietic-cells.Blood. 1992; 80: 3044-3050Crossref PubMed Google Scholar). However, this observation provides no insights into the composition, cellular identity, or differentiation potential of the LS−K population. Thus, the functional and phenotypic significance of the loss of Sca-1 expression was explored. The ability to perform relative quantification proteomics analyses on flow cytometrically enriched primitive hematopoietic cell populations offers opportunities for analysis of transcription changes versus proteome changes. LS+K cells have a more primitive phenotype than LS−K cells using the above assays yet are poorly defined in terms of developmental potential. Transcriptome and proteome analyses were performed to determine the potential cellular fate of LS−K cells and identify whether post-translational regulation of protein expression is observed in LS+K to LS−K transition. Isobaric tagging of tryptic digests from cell populations followed by two-dimensional liquid chromatography and tandem mass spectrometry was applied to LS+K and LS−K cells (see Fig. 1B for workflow). In the first instance a high level of reproducibility in this technique was demonstrated by comparing two separate preparations of LS−K cells with each other (Fig. 2A). Different preparations of LS−K cells showed -fold differences in expression between 0.8 and 1.2 for the vast majority of proteins. The same experiment performed for two separate preparations of LS+K cells also showed marked similarity between two preparations of these cells (Fig. 2B). Thus, the reproducibility of this approach for biological replicates of sorted primitive hematopoietic cell populations was confirmed, and any differences lying outside this 0.8–1.2-fold difference boundary in comparisons of two distinct populations can be considered potentially significant. Three such comparisons were then performed between LS−K and LS+K cells. A representative experiment is shown in Fig. 2C. It can be seen that there are profound differences occurring that the control experiments discussed above demonstrated are not due to biological or experimental variability. Where significant differences were seen, MS/MS spectra were manually checked to validate sequence assignment and reporter group relative quantification. An example of the raw MS data from some key protein changes are provided in supplemental Table 2 with appropriate example spectra in supplemental Fig. 1. LS+K and LS−K cells have profoundly different proteomes even though the populations are separated only on this single marker. In total relative quantification on over 1263 proteins (with >3 peptides quantified) was achieved with starting material of the order of 90 μg of protein. There were 96 proteins showing a 1.2-fold or greater increase and 121 proteins with a 0.83-fold or greater decrease in expression. The total number of proteins found in a single analysis only yet meeting the above criteria was 20 up-regulated and nine down-regulated proteins. A full list of proteins detected including those showing a significant change is shown in supplemental Table 1. The next question we addressed was whether the LS+K to LS−K transition at the proteome level is congruent with changes seen at the transcriptome level. We perfor" @default.
• W2143869444 created "2016-06-24" @default.
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• W2143869444 date "2008-03-01" @default.
• W2143869444 modified "2023-10-09" @default.
• W2143869444 title "Developmental Fate Determination and Marker Discovery in Hematopoietic Stem Cell Biology Using Proteomic Fingerprinting" @default.
• W2143869444 cites W1557986998 @default.
• W2143869444 cites W1578126435 @default.
• W2143869444 cites W1588840730 @default.
• W2143869444 cites W1932806396 @default.
• W2143869444 cites W1964749595 @default.
• W2143869444 cites W1966177375 @default.
• W2143869444 cites W1969920722 @default.
• W2143869444 cites W1973035870 @default.
• W2143869444 cites W1987007640 @default.
• W2143869444 cites W1992665817 @default.
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• W2143869444 cites W2021276131 @default.
• W2143869444 cites W2023544072 @default.
• W2143869444 cites W2030265255 @default.
• W2143869444 cites W2030794327 @default.
• W2143869444 cites W2037695813 @default.
• W2143869444 cites W2051733901 @default.
• W2143869444 cites W2062896090 @default.
• W2143869444 cites W2074218681 @default.
• W2143869444 cites W2082927361 @default.
• W2143869444 cites W2083632015 @default.
• W2143869444 cites W2084080535 @default.
• W2143869444 cites W2090786449 @default.
• W2143869444 cites W2091253171 @default.
• W2143869444 cites W2100378064 @default.
• W2143869444 cites W2121464784 @default.
• W2143869444 cites W2129541995 @default.
• W2143869444 cites W2137956382 @default.
• W2143869444 cites W2139285351 @default.
• W2143869444 cites W2140922568 @default.
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• W2143869444 cites W2165862139 @default.
• W2143869444 cites W2166830325 @default.
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