Human CD34+ CD133+ Hematopoietic Stem Cells Cultured with Growth Factors Including Angptl5 Efficiently Engraft Adult NOD-SCID Il2rγ−/− (NSG) Mice
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{"title"=>"Human CD34+ CD133+ hematopoietic stem cells cultured with growth factors including Angptl5 efficiently engraft adult NOD-SCID il2rγ-/- (NSG) mice", "type"=>"journal", "authors"=>[{"first_name"=>"Adam C.", "last_name"=>"Drake", "scopus_author_id"=>"54919668000"}, {"first_name"=>"Maroun", "last_name"=>"Khoury", "scopus_author_id"=>"8334700500"}, {"first_name"=>"Ilya", "last_name"=>"Leskov", "scopus_author_id"=>"6603479569"}, {"first_name"=>"Bettina P.", "last_name"=>"Iliopoulou", "scopus_author_id"=>"23767182800"}, {"first_name"=>"Maria", "last_name"=>"Fragoso", "scopus_author_id"=>"36828445600"}, {"first_name"=>"Harvey", "last_name"=>"Lodish", "scopus_author_id"=>"36050903500"}, {"first_name"=>"Jianzhu", "last_name"=>"Chen", "scopus_author_id"=>"8148835500"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"sgr"=>"79955723192", "doi"=>"10.1371/journal.pone.0018382", "pui"=>"361724272", "pmid"=>"21559522", "scopus"=>"2-s2.0-79955723192", "issn"=>"19326203", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)"}, "id"=>"eb76ab91-3749-386f-955c-8d9cd15566d5", "abstract"=>"Increasing demand for human hematopoietic stem cells (HSCs) in clinical and research applications necessitates expansion of HSCs in vitro. Before these cells can be used they must be carefully evaluated to assess their stem cell activity. Here, we expanded cord blood CD34(+) CD133(+) cells in a defined medium containing angiopoietin like 5 and insulin-like growth factor binding protein 2 and evaluated the cells for stem cell activity in NOD-SCID Il2rg(-/-) (NSG) mice by multi-lineage engraftment, long term reconstitution, limiting dilution and serial reconstitution. The phenotype of expanded cells was characterized by flow cytometry during the course of expansion and following engraftment in mice. We show that the SCID repopulating activity resides in the CD34(+) CD133(+) fraction of expanded cells and that CD34(+) CD133(+) cell number correlates with SCID repopulating activity before and after culture. The expanded cells mediate long-term hematopoiesis and serial reconstitution in NSG mice. Furthermore, they efficiently reconstitute not only neonate but also adult NSG recipients, generating human blood cell populations similar to those reported in mice reconstituted with uncultured human HSCs. These findings suggest an expansion of long term HSCs in our culture and show that expression of CD34 and CD133 serves as a marker for HSC activity in human cord blood cell cultures. The ability to expand human HSCs in vitro should facilitate clinical use of HSCs and large-scale construction of humanized mice from the same donor for research applications.", "link"=>"http://www.mendeley.com/research/human-cd34-cd133-hematopoietic-stem-cells-cultured-growth-factors-including-angptl5-efficiently-engr", "reader_count"=>94, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>4, "Researcher"=>28, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>27, "Student > Postgraduate"=>4, "Student > Master"=>14, "Other"=>3, "Student > Bachelor"=>3, "Lecturer"=>2, "Professor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>4, "Researcher"=>28, "Student > Doctoral Student"=>4, "Student > Ph. D. Student"=>27, "Student > Postgraduate"=>4, "Student > Master"=>14, "Other"=>3, "Student > Bachelor"=>3, "Lecturer"=>2, "Professor"=>3}, "reader_count_by_subject_area"=>{"Engineering"=>2, "Unspecified"=>4, "Biochemistry, Genetics and Molecular Biology"=>9, "Nursing and Health Professions"=>1, "Agricultural and Biological Sciences"=>58, "Medicine and Dentistry"=>16, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Immunology and Microbiology"=>3}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>2}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>16}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>3}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>58}, "Nursing and Health Professions"=>{"Nursing and Health Professions"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>9}, "Unspecified"=>{"Unspecified"=>4}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}}, "reader_count_by_country"=>{"Republic of Singapore"=>1, "United States"=>4, "United Kingdom"=>1, "Germany"=>1}, "group_count"=>6}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/778208"], "description"=>"<p>2.5–5×10<sup>5</sup> expanded CD34<sup>+</sup> CD133<sup>+</sup> cells were transferred into the primary neonate or adult NSG recipients. 14–28 weeks after transfer, single cell suspension was prepared from the bone marrow of recipient mice. Human cells were isolated by either depleting mouse CD45<sup>+</sup> cells or enriching for human CD34<sup>+</sup> cells and then transferred into secondary neonate or adult recipients. Each secondary recipient received an equivalent of one femur's worth of enriched human cells. The numbers of CD34<sup>+</sup> cells were calculated and shown to the nearest 10,000 cells. n.d. – not determined. 12 weeks after secondary transfer, chimerism was assessed in the bone marrow by staining for human and mouse CD45.</p>", "links"=>[], "tags"=>["reconstitutions", "nsg"], "article_id"=>448573, "categories"=>["Cell Biology", "Developmental Biology", "Immunology"], "users"=>["Adam C. Drake", "Maroun Khoury", "Ilya Leskov", "Bettina P. Iliopoulou", "Maria Fragoso", "Harvey Lodish", "Jianzhu Chen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0018382.t001", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_secondary_reconstitutions_in_NSG_mice_/448573", "title"=>"Summary of secondary reconstitutions in NSG mice.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 18:42:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/777981"], "description"=>"<p>(A) Comparison of chimerism in peripheral blood at 3–4 and 6–7 months post reconstitution. Sub lethally irradiated adult NSG mice were injected with CD34<sup>+</sup> CD133<sup>+</sup> cells (2.5–5×10<sup>5</sup> per mouse) 3–4 and 6–7 months post injection PBMCs were stained for human and mouse CD45. Mean ± SD is shown with 6 mice per group. (B) Presence of human CD34<sup>+</sup> CD133<sup>+</sup> cells in the bone marrow of reconstituted mice. Cells from the bone marrow of primary recipient mice 3–4 month after reconstitution were analyzed for human CD45, CD34 and CD133. Representative CD34 versus CD133 staining profiles are shown for human CD45<sup>+</sup> cells. The number indicates the percentage of cells in the gated region. Serial reconstitution. Single cell suspension was prepared from the bone marrow of the primary recipient mice 14–28 weeks after engraftment. Human cells were enriched by depleting cells expressing mouse CD45 or by positive selection for human CD34<sup>+</sup> cells by magnetic sorting. An equivalent of one femur's worth of enriched human cells was transferred into one sub-lethally irradiated secondary neonate or adult recipient. Twelve weeks later, cells from the spleen and bone marrow of secondary recipient mice were analyzed for human and mouse CD45. Human CD45 versus mouse CD45 staining profiles are shown for the secondary mouse with the highest human chimerism (n = 10, see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018382#pone-0018382-t001\" target=\"_blank\">Table 1</a>).</p>", "links"=>[], "tags"=>["cells"], "article_id"=>448349, "categories"=>["Cell Biology", "Developmental Biology", "Immunology"], "users"=>["Adam C. Drake", "Maroun Khoury", "Ilya Leskov", "Bettina P. Iliopoulou", "Maria Fragoso", "Harvey Lodish", "Jianzhu Chen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0018382.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expanded_CD34_CD133_cells_contain_long_term_HSCs_/448349", "title"=>"Expanded CD34<sup>+</sup> CD133<sup>+</sup> cells contain long-term HSCs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:40:53"}
  • {"files"=>["https://ndownloader.figshare.com/files/778041"], "description"=>"<p>(A) Comparison of reconstitution levels in mice injected with different numbers of expanded cells. Sub-lethally irradiated adult NSG recipients were injected with cultured cells containing 0.5×10<sup>5</sup>, 2.5×10<sup>5</sup> or 5×10<sup>5</sup> CD34<sup>+</sup> CD133<sup>+</sup> cells. Eight weeks later, PBMCs were analyzed for human and mouse CD45 expression. Mean ± SD is shown, n = 6–8 per group. (B) Comparison of reconstitution levels in the bone marrow of neonate and adult recipients. Sub-lethally irradiated adult NSG recipients were injected with expanded cells containing 2.5×10<sup>5</sup> CD34<sup>+</sup> CD133<sup>+</sup> cells. Sub-lethally irradiated neonates were injected with 1×10<sup>5</sup> unexpanded or expanded CD34<sup>+</sup> CD133<sup>+</sup> cells. Twelve to sixteen week later, bone marrow was analyzed for human and mouse CD45 expression. Mean ± SD is shown, n = 4–6 per group.</p>", "links"=>[], "tags"=>["cells", "robust", "reconstitution", "neonate"], "article_id"=>448408, "categories"=>["Cell Biology", "Developmental Biology", "Immunology"], "users"=>["Adam C. Drake", "Maroun Khoury", "Ilya Leskov", "Bettina P. Iliopoulou", "Maria Fragoso", "Harvey Lodish", "Jianzhu Chen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0018382.g003", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expanded_CD34_CD133_cells_give_robust_reconstitution_in_both_neonate_and_adult_recipients_/448408", "title"=>"Expanded CD34<sup>+</sup> CD133<sup>+</sup> cells give robust reconstitution in both neonate and adult recipients.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:41:09"}
  • {"files"=>["https://ndownloader.figshare.com/files/778113"], "description"=>"<p>Sub-lethally irradiated NSG adults were injected with 2.5–5×10<sup>5</sup> expanded CD34<sup>+</sup> CD133<sup>+</sup> cells. Cells in the bone marrow (A–E, L), blood (F, O), thymus (M) and spleen (G–J, K, N) were analyzed by flow cytometry 12 weeks after reconstitution. Shows are staining profiles for erythrocytes (CD235ab<sup>+</sup>) and platelets (CD41<sup>+</sup>) (A and F), total leukocytes (human CD45<sup>+</sup>) (B and G), myelomonocytes (CD14<sup>+</sup> CD33<sup>+</sup>) (C and H), dendritic cells (CD11c<sup>+</sup> CD209<sup>+</sup>) (D and I), granulocytes (CD15<sup>+</sup>) and natural killer cells (CD56<sup>+</sup>) (E and J), B cell stages (proB - CD34<sup>+</sup> CD10<sup>+</sup>, immature - CD10<sup>+</sup>, mature - IgM<sup>+</sup> IgD<sup>+</sup>) (K and L), T cell precursors (double negative, double positive and single positive) (M) and mature T cells (CD45<sup>+</sup> CD3<sup>+</sup> expressing either CD4 or CD8) (N and O). The numbers indicate percentages of cells in the gated region. Representative data from one of at least 5 mice are shown. All cells shown are gated on live human cells (FSC/SSC/live/CD45<sup>+</sup>) except A, F (FSC/SSC only) B, G (FSC/SSC/live) and the IgM vs IgG stains (FSC/SSC/live/CD45<sup>+</sup>/CD19<sup>+</sup>). Summaries of human cell characterization can also be found in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018382#pone-0018382-t002\" target=\"_blank\">Table 2</a>.</p>", "links"=>[], "tags"=>["reconstitution", "nsg"], "article_id"=>448479, "categories"=>["Cell Biology", "Developmental Biology", "Immunology"], "users"=>["Adam C. Drake", "Maroun Khoury", "Ilya Leskov", "Bettina P. Iliopoulou", "Maria Fragoso", "Harvey Lodish", "Jianzhu Chen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0018382.g004", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Multi_lineage_reconstitution_in_adult_NSG_mice_/448479", "title"=>"Multi-lineage reconstitution in adult NSG mice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:41:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/777848"], "description"=>"<p>(A) Limiting dilution analysis comparing cultured (squares) vs. uncultured (circles) cells for SRC frequency. CD133<sup>+</sup> cord blood cells (5,000, 1,000 and 400) and their cultured progeny were injected into sub-lethally irradiated neonate recipients. Recipients were analyzed for human cell reconstitution in the bone marrow by staining for human CD45 and mouse CD45 followed by flow cytometry. SRC frequencies calculated by Poisson statistics are shown for each population based on number of input CD34<sup>+</sup> CD133<sup>+</sup> cells. Values for a 0.5% cutoff (top) and 0.1% cutoff (bottom) are shown. (B) Representative CD34 versus CD133 staining profiles of purified human HSCs at the start of in vitro expansion and cells after culture for 10 days. The number indicates percentage of CD34<sup>+</sup> CD133<sup>+</sup> cells in the gated region. (C) Comparison of bone marrow chimerism (human CD45 vs. mouse CD45.1) in sub-lethally irradiated neonate NSG recipients injected with 5000, 1000 and 400 cultured or uncultured CD34<sup>+</sup> CD133<sup>+</sup> cells. Horizontal bars show the mean, each symbol is an individual mouse. Two tail ANOVA testing shows no significant differences between data sets (p = 0.23). (D) Comparison of reconstitution in neonate NSG recipients transferred with 100,000 purified, cultured CD34<sup>+</sup> CD133<sup>+</sup> cells or total cultured cells (unpurified) containing 100,000 CD34<sup>+</sup> CD133<sup>+</sup> cells. Peripheral blood mononuclear cells were analyzed for human and mouse CD45 12 weeks after transfer. Mean ± SD is shown with 7 mice per group. (E) Limiting dilution analysis comparing purified (circles) and unpurified (squares) CD34<sup>+</sup> CD133<sup>+</sup> cells for SRC frequency. 300 and 1000 purified CD34<sup>+</sup> CD133<sup>+</sup> cells or total cells containing 300 and 1000 CD34<sup>+</sup> CD133<sup>+</sup> cells were transferred into sub-lethally irradiated adult NSG mice. Recipients were analyzed for human cell reconstitution in the bone marrow 8 weeks later by flow cytometry. Frequencies calculated using Poisson statistics are shown for each population based on the number of CD34<sup>+</sup> CD133<sup>+</sup> cells. All limiting dilutions represent 6–10 mice per data point and are single cultures representative of at least 2 independent experiments.</p>", "links"=>[], "tags"=>["cells", "scid", "repopulating"], "article_id"=>448210, "categories"=>["Cell Biology", "Developmental Biology", "Immunology"], "users"=>["Adam C. Drake", "Maroun Khoury", "Ilya Leskov", "Bettina P. Iliopoulou", "Maria Fragoso", "Harvey Lodish", "Jianzhu Chen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0018382.g001", "stats"=>{"downloads"=>1, "page_views"=>13, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expanded_CD34_CD133_cells_are_SCID_repopulating_cells_/448210", "title"=>"Expanded CD34<sup>+</sup> CD133<sup>+</sup> cells are SCID repopulating cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-20 18:40:07"}
  • {"files"=>["https://ndownloader.figshare.com/files/778245"], "description"=>"<p>Data are a compilation of 24 mice. Each stain was performed on a minimum of 4 mice and all stains were performed on mice derived from at least two unrelated cords. All percentage calculations are based on the following gated groups: human leukocytes on total live (DAPI/PI negative) human or mouse CD45<sup>+</sup> cells, erythrocytes and platelets on FSC versus SSC gate, and all other groups on live human CD45<sup>+</sup> cells. Shown are mean percentage and SD of specific cell types in blood, spleen and bone marrow. ND, not determined.</p>", "links"=>[], "tags"=>["reconstitution", "nsg", "mice", "injected", "expanded"], "article_id"=>448607, "categories"=>["Cell Biology", "Developmental Biology", "Immunology"], "users"=>["Adam C. Drake", "Maroun Khoury", "Ilya Leskov", "Bettina P. Iliopoulou", "Maria Fragoso", "Harvey Lodish", "Jianzhu Chen"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0018382.t002", "stats"=>{"downloads"=>2, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Summary_of_reconstitution_of_adult_NSG_mice_injected_with_2_5_5_10_5_expanded_CD34_CD133_cells_/448607", "title"=>"Summary of reconstitution of adult NSG mice injected with 2.5–5×10<sup>5</sup> expanded CD34<sup>+</sup>CD133<sup>+</sup>cells.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2013-02-20 18:42:11"}

PMC Usage Stats | Further Information

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