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Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential

Rita C. R. Perlingeiro^1,*, Michael Kyba^1,* and George Q. Daley^2,

¹ Whitehead Institute, 9 Cambridge Center, Cambridge, MA 02142, USA
² Division of Hematology/Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02115, USA
^* These authors contributed equally to this work

View larger version (25K): [in a new window]   Fig. 1. Strategy for retroviral expression of BCR/ABL in differentiated ES cells to target and expand hematopoietic progenitors. A-D refer to panels in Fig. 2.

View larger version (48K): [in a new window]   Fig. 2. Growth and hematopoietic potential of BCR/ABL-infected EB cells. (A) Methylcellulose colony formation of BCR/ABL-infected EB cells after 8 days on OP9 cells under several culture conditions. BL-MCM represents MCM containing SCF, VEGF and D4T-conditioned medium; Myelo-MCM represents MCM containing IL3, IL6, SCF and Epo; Base-MCM represents MCM without growth factors. (B) Growth of a representative liquid culture of BCR/ABL (bulk) and empty vector-infected EB cells in the presence of IL3, IL6 and SCF after 8 days on OP9 cells. (C) Colony formation of bulk cells after 30 days in liquid culture (without stroma). (D) Colony formation of clonal cells derived from BCR/ABL bulk cells. (E) Morphology of blast colonies (BL-CFC) (left) and corresponding cell populations (right). (F) Morphology of a primitive erythroid colony (EryP) (left) and corresponding nucleated primitive erythroid cells (right). Cytospins were stained with May-Grunwald-Giemsa. Experiments A, C and D were performed two, four and five times, respectively.

View larger version (49K): [in a new window]   Fig. 3. Engraftment of adult mice. Frequency of GFP-positive cells in bone marrow (BM), peripheral blood (PB) and spleen (Sp) of mice injected with bulk cells (A) or clonal cells (B). (C) Antibody staining profile of GFP-positive cells for Gr1/Mac1, Ter119, B220 and CD4/CD8 in BM, PB and Sp of mice injected with bulk cells. (D) Antibody staining profile of GFP-positive cells for Gr1/Mac1, Ter119, B220 and CD4/CD8 in BM, PB and Sp of mice injected with clonal cells. The number of mice engrafted and the total number of mice injected are indicated, respectively, in parentheses. (E) GFP-positive cells from peripheral blood were stained with lineage-specific antibodies, sorted, centrifuged onto glass slides and stained with May-Grunwald-Giemsa.

View larger version (46K): [in a new window]   Fig. 4. Flow cytometric analysis and globin gene expression of clones. (A) FACS-staining profile of clonal cells maintained in liquid culture in vitro. (B) RT-PCR analysis for adult (ß-major) and embryonic (ß-H1) globin mRNA from clones derived from BCR/ABL bulk cells (E1, E2 and E3) and sorted cells (GFP-positive Ter119-positive) from spleen (Sp) and bone marrow (BM) of mice injected with clones E1 and E2, respectively. A threefold dilution series for each sample confirmed that the PCR was in the linear range.

View larger version (114K): [in a new window]   Fig. 5. Hematopoietic colonies derived from the bone marrow (BM) of engrafted mice. Bone marrow from a primary engrafted animal was harvested and plated in MCM supplemented with growth factors (IL3, IL6, SCF and Epo). After 7-10 days, GFP-positive colonies were observed by fluorescence microscopy (A,C). Colonies were plucked, disrupted, centrifuged onto a glass slide, and stained to reveal hematopoietic morphology (B,D). (A,B) CFU-GEMM; (C,D) CFU-GM.

View larger version (27K): [in a new window]   Fig. 6. Schematic representation of hematopoietic differentiation in embryos and embryoid bodies. Pluripotent cells (ES cells or cells from the inner cell mass, ICM) undergo mesodermal commitment and differentiation into the common precursor of hematopoietic and endothelial lineages (hemangioblast). We have demonstrated a cell with both primitive erythroid and definitive lymphoid-myeloid potential, properties predicted for the earliest embryonic hematopoietic stem cell: -HSC. The -HSC is deficient at repopulation of adult hosts owing to some combination of its transient nature, its homing properties and its response to the adult environment (top panel). BCR/ABL (lower panel) enables repopulation by altering one or more of these deficiencies, allowing both lymphoid and myeloid potential to be observed in engrafted animals.