Radiation Research

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Radiation Research 166(2):345-351. 2006
doi: 10.1667/RR3595.1

Regeneration of Megakaryocytopoiesis and Thrombopoiesis In Vitro from X-Irradiated Human Hematopoietic Stem Cells

Ikuo Kashiwakura1a, Osamu Inanamib, Yoshinao Abec, Kei Satohd, Tsuneo A. Takahashie, and Mikinori Kuwabarab

aDepartment of Radiological Technology, Hirosaki University School of Health Sciences, 66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan;

bLaboratory of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18, Nishi-9, Kita-ku, Sapporo 060-0818, Japan

cLaboratory of Radiology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan

dDepartment of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan

eCell Processing Department, Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

11Address for correspondence: Department of Radiological Technology, Hirosaki University School of Health Sciences, 66-1 Hon-cho, Hirosaki 036–8564, Japan;

Abstract

Kashiwakura, I., Inanami, O., Abe Y., Satoh, K., Takahashi, T. A. and Kuwabara, M. Regeneration of Megakaryocytopoiesis and Thrombopoiesis In Vitro from X-Irradiated Human Hematopoietic Stem Cells. Radiat. Res. 166, 345–351 (2006).

In the present study, we investigated whether X-irradiated hematopoietic stem cells can be induced to undergo megakaryocytopoiesis and thrombopoiesis in vitro using cytokine combinations that have been demonstrated to be effective for conferring increased survival on irradiated human CD34+ megakaryocytic progenitor cells (colony-forming unit megakaryocytes; CFU-Meg), such as thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor and FLT3 ligand. Culture of nonirradiated CD34+ cells in serum-free medium supplemented with multiple cytokine combinations led to an approximately 200- to 600-fold increase in the total cell numbers by day 14 of culture. In contrast, the growth of X-irradiated cells was observed to be one-sixth to one-tenth that of the nonirradiated cultures. Similarly, total megakaryocytes were increased by 50- to 130-fold, while culture of X-irradiated cells yielded one-fourth to one-eighth of the control numbers. At this time, CD41+ particles, which appeared to be platelets, were produced in the medium harvested from nonirradiated and irradiated cultures. Although radiation suppressed cell growth and megakaryocytopoiesis, there were no significant differences in thrombopoiesis between the two types of culture. These results suggest that X-irradiated CD34+ cells can be induced to undergo nearly normal terminal maturation through megakaryocytopoiesis and thrombopoiesis by stimulation with appropriate cytokine combinations.

Received: April 27, 2005; Accepted: April 10, 2006



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FIG. 1. Total numbers of mononuclear cells produced in liquid culture. Freshly nonirradiated (0 Gy) and X-irradiated (2 Gy) CD34+ cells (4 × 103 cells/ml) were cultured in serum-free medium supplemented with the cytokine combinations shown in the figure. On day 14 of culture, cells were harvested and the numbers of viable cells were determined by trypan blue exclusion. Values are means ± SD of three separate experiments, each performed in three wells. a, P < 0.001 and P < 0.05 for 3ST (IL3 + SCF + TPO) compared to 3T (IL3 + TPO) and 3S (IL3 + SCF). b, P < 0.01 and P < 0.001 for 3SFT (IL3 + SCF + FL + TPO) compared to 3T, 3S and ST (SCF + TPO). c, P < 0.001 and P < 0.05 for 3ST or 3SFT compared to 3T and ST, 3S.

FIG. 2. Results of flow cytometry analysis of cells harvested from cultures stimulated with the various cytokine combinations on day 14 of culture. The cells were treated with anti-human FITC-CD34, PE-CD41 and PC5-CD45 monoclonal antibodies. The expression of each surface antigen was analyzed by flow cytometry. 3T: IL3 + TPO; ST: SCF + TPO; 3S: IL3 + SCF; 3ST: IL3 + SCF + TPO; 3SFT: IL3 + SCF + FL + TPO. Each value is the mean ± SD of four to five separate experiments. a, P < 0.05 for 3SFT compared to 3T, ST and 3S. b, P < 0.01 for 3SFT compared to each of the other combinations. c, P < 0.01 for 3T(0 Gy), ST(0 Gy) and ST(2 Gy) each compared with 3S, 3ST and 3SFT. d, P < 0.01 for 3SFT compared to each of the other combinations.

FIG. 3. Total numbers of megakaryocytes in liquid cultures. Freshly prepared nonirradiated (0 Gy) and X-irradiated (2 Gy) CD34+ cells were cultured in serum-free medium supplemented with the cytokine combinations listed in the figure. On day 14 of culture, cells were harvested and the expression of CD41+CD45+ was analyzed by flow cytometry. The total numbers of megakaryocytes were calculated from the total number of cells harvested from the culture and the proportions of CD41+CD45+ cells. The initial number of megakaryocytes was 236 ± 68. Values are the means ± SD of three separate experiments, each performed in three wells. a, P < 0.05 for ST was compared to 3S.

FIG. 4. Total numbers of CFU-Meg generated in liquid culture. Nonirradiated (0 Gy) and X-irradiated (2 Gy) CD34+ cells were cultured in serum-free medium supplemented with the cytokine combinations listed in the figure. On day 14, the cells harvested from each culture were assayed for the numbers of CFU-Meg using a plasma clot culture supplemented with TPO + SCF. The total numbers of CFU-Meg were calculated from the total numbers of cells harvested and the number of colonies per well. The initial numbers of CFU-Meg were 122 ± 76 (0 Gy) and 24 ± 5 (2 Gy), respectively. Values are the means ± SD of three separate experiments, each performed in three wells. a, P < 0.05 and P < 0.01 for 3ST compared to each of 3T and 3S.

FIG. 5. Estimation of thrombopoiesis in cultures of nonirradiated and X-irradiated CD34+ cells. Nonirradiated (0 Gy) and X-irradiated (2 Gy) CD34+ cells were cultured in serum-free medium supplemented with the cytokine combinations listed in the figure. On day 14, each medium was harvested and the number of platelet-like CD41+-positive particles was analyzed by flow cytometry. Values are the means ± SD of three separate experiments. a, P < 0.001 for 3T compared with each of the other combinations.

FIG. 6. Results of flow cytometry analysis for the markers of platelet activation. Platelets were harvested from cultures stimulated with the combination of IL3 + TPO on day 14 of culture. The platelets were treated with anti-human PE-CD62P and FITC-PAC1 monoclonal antibodies. The expression of each surface antigen was analyzed by flow cytometry. Each value shows the percentage of CD62P- and PAC1-positive cells gated in the cellular region. Typical cytograms are shown for three separate experiments.

Cited by

Atsuko Omori, Kenji Takahashi, Masaharu Hazawa, Naoko Misaki, Hisateru Ohba, Mami Manabe, Haruo Sato, Kaori Kudo, Tsuneo A. Takahashi, Ikuo Kashiwakura. (2008) Maternal and Neonatal Factors Associated with the High Yield of Mononuclear Low-Density/CD34+ Cells from Placental/Umbilical Cord Blood. The Tohoku Journal of Experimental Medicine 215:1, 23-32
Online publication date: 1-Feb-2008.
CrossRef
Kenji Takahashi, Satoru Monzen, Kiyomi Eguchi-Kasai, Yoshinao Abe, Ikuo Kashiwakura. (2007) Severe Damage of Human Megakaryocytopoiesis and Thrombopoiesis by Heavy-Ion Beam Radiation. Radiation Research 168:5, 545-551
Online publication date: 1-Nov-2007.

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