Human hematopoietic stem cells can survive in vitro for several months

Abstract

We previously reported that long-lasting in vitro hematopoiesis could be achieved using the cells differentiated from primate embryonic stem (ES) cells. Thus, we speculated that hematopoietic stem cells differentiated from ES cells could sustain long-lasting in vitro hematopoiesis. To test this hypothesis, we investigated whether human hematopoietic stem cells could similarly sustain long-lasting in vitro hematopoiesis in the same culture system. Although the results varied between experiments, presumably due to differences in the quality of each hematopoietic stem cell sample, long-lasting in vitro hematopoiesis was observed to last up to nine months. Furthermore, an in vivo analysis in which cultured cells were transplanted into immunodeficient mice indicated that even after several months of culture, hematopoietic stem cells were still present in the cultured cells. To the best of our knowledge, this is the first report to show that human hematopoietic stem cells can survive in vitro for several months.

Figure 1

Appearance of the cells attached to feeder cells. Representative examples of CD34(+) human hematopoietic stem/progenitor cells cultured on either OP9 feeder cells ((a), (c)) or C3H10T1/2 feeder cells ((b), (d)) for 7 days ((a), (b)) or 22 days ((c), (d)).

Figure 2

Production of hematopoietic cells in long-term cultures of human hematopoietic stem cells. (a) Eight independent experiments were performed using eight different umbilical cord blood samples and OP9 cells as feeder cells. (b) Seven independent experiments were performed using seven different umbilical cord blood samples and C3H10T1/2 cells as feeder cells. ((a), (b)) The number of detached cells in the overlying medium was counted at each medium change (approximately half weekly). The data are shown as the mean number of detached cells produced from a single CD34(+) cell, that is, the total number of detached cells divided by the number of CD34(+) cells used to initiate the culture. Exp: experiment. A to I after Exp-OP9 and Exp-10T1/2 indicate 9 different umbilical cord blood samples derived from 9 different neonates. #: Cultures Exp-OP9-A and Exp-10T1/2-F were terminated because of fungal infection.

Figure 3

Characterization of cultured cells. (a) Flow cytometric analysis of detached cells produced in cultures on OP9 (Exp-OP9-A) and C3H10T1/2 (Exp-10T1/2-A) feeder cells and collected on Day 218 of culture. The detached cells were stained for CD33, a marker specific for granulocyte/macrophage lineage cells, and CD34, a marker specific for hematopoietic stem/progenitor cells. Flow cytometric analyses of detached cells from other experiments showed similar results. (b) Colony-formation assays. Detached cells produced in culture on OP9 feeder cells (Exp-OP9-A) were collected on Days 57 and 127 of culture. Similarly, detached cells produced in culture on C3H10T1/2 feeder cells (Exp-10T1/2-D) were collected on Days 60 and 134 of culture. The cell samples were used in a standard colony-formation assay. Black bars: colony-forming unit of monocyte/macrophage lineage cells, CFU-M. Blue bars: colony-forming unit of granulocyte lineage cells, CFU-G. Yellow bars: colony-forming unit of granulocyte and monocyte/macrophage lineage cells, CFU-GM. Red bars: burst-forming unit of erythroid cells, BFU-E. Similar results were obtained in colony-formation assays using detached cells from other cultures.

Figure 4

Flow cytometric analysis of hematopoietic cells of the mouse that had been transplanted with human hematopoietic cells produced by in vitro culture. ((a), (e)) Schema of the experimental procedure and flow cytometric analysis of transplanted cells. ((a)–(d)) Detached cells (3.9 × 10⁶ cells) produced on OP9 feeder cells (Exp-OP9-A) were collected on Day 169 of culture and transplanted into an immunodeficient NOG mouse. Peripheral blood was collected on Days 56 (b) and 112 (c) after transplantation, and bone marrow cells were collected on Day 126 after transplantation (d). ((e), (f)) Detached cells (2.4 × 10⁶ cells) produced on OP9 feeder cells (Exp-OP9-F) were collected on day 125 of culture and transplanted into an immunodeficient NOG mouse. The bone marrow cells were collected on Day 184 after transplantation and were analyzed. ((a)–(f)) The cells were stained using monoclonal antibodies against CD45, a leukocyte common antigen, CD34, a marker specific for hematopoietic stem/progenitor cells, CD33 and CD13, markers of granulocyte and monocyte/macrophage lineage cells, CD11b and CD14, markers of monocyte/macrophage lineage cells, CD19, a marker of B lymphocyte lineage cells, CD3, CD4, and CD8, markers of T lymphocyte lineage cells, Gly-A (Glycophorin A), a marker of erythroid cells, CD56, a marker of large granular lymphocytes and natural killer cells, and CD41a, a marker of megakaryocyte/platelet lineage cells.