1. Emergence of muscle and neural hematopoiesis in humans
by Karen E. Jay, Lisa Gallacher, and Mickie Bhatia
Blood
Volume 100(9):
November 1, 2002
©2002 by American Society of Hematology

2. Flow cytometric analysis of cell surface phenotype of cells derived from human muscle and neural tissues.Flow cytometry was used to evaluate the cell surface expression of the human-specific proteins associated with hematopoietic tissue: AC133, and cell dif...
Flow cytometric analysis of cell surface phenotype of cells derived from human muscle and neural tissues.Flow cytometry was used to evaluate the cell surface expression of the human-specific proteins associated with hematopoietic tissue: AC133, and cell differentiation markers CD34 and CD45. A representative FACS analysis of de novo–isolated muscle (A) and neural (B) cells is shown, and the percentage of subpopulations expressing single or coexpressing human hematopoietic markers is shown as the average mean ± SEM (n = 4) in each quadrant. Specificity of hematopoietic antibody staining and background signal was determined by comparing muscle and neural cells stained with mouse IgG1 (Ai and Bi) to establish positive quadrant levels to omit fluorescence because of nonspecific binding and auto fluorescence properties of the cells. Results are based on data from 4 independent samples of muscle and neural tissues analyzed in duplicate.
Karen E. Jay et al. Blood 2002;100:
©2002 by American Society of Hematology

3. Human chimerism in the tissue of immune-deficient mice that received intravenous transplants of human fetal tissues.Comparison of human hematopoietic chimerism from hematopoietic and nonhematopoietic sources.
Human chimerism in the tissue of immune-deficient mice that received intravenous transplants of human fetal tissues.Comparison of human hematopoietic chimerism from hematopoietic and nonhematopoietic sources. Scatterplots show FACS analysis of mouse BM stained with the pan-leukocyte marker CD45 for the detection of human hematopoietic cells (gated box) after transplantation with isotype control (A), fetal blood (B), fetal liver (C), fetal BM (D), fetal muscle (E), and fetal neural cells (F). Cell doses ranged between 2 × 106 and 5 × 106 to up to 15 × 106 for muscle and neural transplants (n = 43).
Karen E. Jay et al. Blood 2002;100:
©2002 by American Society of Hematology

4. Immunohistochemical and morphologic analysis of human fetal muscle and neural cells using tissue-specific markers and emergence of functional hematopoietic progenitors.Light microscopy was used to visualize cells comprising human muscle (Ai) and neural (Bi)...
Immunohistochemical and morphologic analysis of human fetal muscle and neural cells using tissue-specific markers and emergence of functional hematopoietic progenitors.Light microscopy was used to visualize cells comprising human muscle (Ai) and neural (Bi) tissues, showing morphologic features associated with these tissue types. Human muscle tissue specificity was analyzed by immunohistochemistry for the expression of muscle-specific markers recognizing heavy chain of myosin (Aii) and the nuclear DNA binding factor, myogenin (Aiii), whereas human neural tissue demonstrated expression of neural progenitor-specific marker, nestin (Bii) and MAP-2 (Biii), respectively. A representative panel of multilineage hematopoietic colonies derived from muscle (Aiv) and neural (Biv) tissues cultured in the presence of HGF together with BMP-4 and EPO is shown. Human hematopoietic colony types generated from muscle and neural tissues include erythroid burst-forming unit (BFU-E), CFU-granulocyte (G), -macrophage (M), and tetrapotent mixed colonies (granulocyte, erythroid, macrophage, megakaryocyte [GEMM]). Similar results were obtained from 9 independent samples of muscle and neural tissue samples. Magnification × 200.
Karen E. Jay et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Evaluation of human hematopoietic cell fate potential of de novo and cultured muscle and neural cells.Primary human muscle (A) and neural (B) tissues were examined for hematopoietic progenitor capacity (CFU) at isolation (day 0) or after 5 days of in vitro ...
Evaluation of human hematopoietic cell fate potential of de novo and cultured muscle and neural cells.Primary human muscle (A) and neural (B) tissues were examined for hematopoietic progenitor capacity (CFU) at isolation (day 0) or after 5 days of in vitro culture in essential media containing 10% CEE, or human hematopoietic growth factors (HGF) alone or with BMP-4 or EPO, as single additions or in combination as indicated. In all culture conditions, media and cytokines were replenished every other day. Single, double, and triple asterisks indicate substantial differences within measured groups of P < .01. Data shown is based on 6 to 13 independent samples analyzed in culture conditions indicated.
Karen E. Jay et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Comparative analysis of human hematopoietic-, muscle-, and neural-derived hematopoietic progenitors.Human tissues indicated were harvested under identical conditions, and functional hematopoietic progenitor capacity was evaluated at day 0 (de novo–isolated ...
Comparative analysis of human hematopoietic-, muscle-, and neural-derived hematopoietic progenitors.Human tissues indicated were harvested under identical conditions, and functional hematopoietic progenitor capacity was evaluated at day 0 (de novo–isolated tissues) and again at day 5 of culture in HGF conditions with or without BMP-4 or BMP-4 together with EPO. (A) Developmental potential of hematopoietic progenitors was compared among various human tissues by enumerating colony types as a measure of hematopoietic lineage commitment that include erythroid (BFU-E), monocytic (CFU-M), granulocytic (CFU-G), and myelocytic (CFU-GM). Composition of CFU types generated for each tissue is expressed as mean percentage ± SEM (n = 5) of the total colonies for muscle (gray), neural (white), and blood (black) cells cultured in essential media containing HGF (i), BMP-4 (ii), or BMP-4 and EPO in combination (iii). Single and double asterisks indicate substantial differences within measured groups ofP < .01, (n = 5). (B) Fold changes in total hematopoietic progenitor expansion was compared in response to culture conditions indicated and compared with day 0 of cultured fetal blood (i), muscle (ii), and neural (iii) cells. Each graph shows the increase in progenitors after culture in essential media containing HGF (●), BMP-4 (♦), or BMP-4 and EPO (▪). (C) Fold expansion of fetal blood cultured for 5 days in the absence or presence of cells derived from fetal muscle (i) or fetal neural (ii) tissue using cytokine combinations indicated. Cells were cocultured using 5.0 × 105 fetal blood cells to equal numbers of fetal muscle or neural cells to maintain the same cell density used in previous experiments shown in panel B. Insets show the relative fold expansion to fetal blood cells cultured alone compared with the various coculture treatments. (D) Hematopoietic progenitors assayed from single cells suspensions of muscle (i), neural (ii), and blood (iii) in the absence or presence of BMP-4 directly added to methylcellulose used in this clonal assay. Graphs show number of clonogenic progenitor of de novo–isolated tissues in the absence of BMP-4 (control) shown standardized as 100% and in the presence of BMP-4 as a percentage relative to control ± SEM. Results are based on a total of 5 independent samples.
Karen E. Jay et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Isolation of AC133+ and AC133−subsets from human embryonic tissues
Isolation of AC133+ and AC133−subsets from human embryonic tissues.Human fetal neural cells were stained with human-specific antibodies raised to the prominin AC133, CD34, and CD45.
Isolation of AC133+ and AC133−subsets from human embryonic tissues.Human fetal neural cells were stained with human-specific antibodies raised to the prominin AC133, CD34, and CD45. AC133+ and AC133− cells were isolated according to sorting gates shown in Figure 1, from the population of cells devoid of CD34 and the hematopoietic marker CD45. (A) Purified subpopulations of AC133+CD34−CD45− (Bi) and AC133−CD45−CD34− (Bii) cells from human neural tissue were cultured under serum-free conditions shown to induce neural hematopoiesis. Magnification × 200 (Bi) and × 400 (Bii). Hematopoietic colonies of multiple lineages were detected from AC133+CD34−CD45− cells. The composition of colonies was similar to that shown in Figure 4. (C) Quantitative analysis of hematopoietic colonies arising from either AC133+ or AC133− subsets from the CD34−CD45− population. Cells were cultured in HGF with BMP-4 and EPO and were then collected and plated into colony-forming assays. Colonies were scored after 12 to 14 days and shown as the average number of colonies per 50 000 cell input ± SEM. Averages shown are based on 4 independent samples.
Karen E. Jay et al. Blood 2002;100:
©2002 by American Society of Hematology