MN-RET induction in the bone marrow cultures in vitro requires terminal erythropoiesis through the continuum of the proliferation and differentiation processes of bone marrow cells. When cultures are studied over prolonged periods of time, this involves critical steps of differentiation from stem cells to progenitor erythroblasts, precursor cells, and RETs. With increasing understanding of the regulation of erythropoiesis, human CD34+ or murine Lin- bone marrow cells are able to be expanded and differentiated along the erythroid lineage to generate RETs and RBCs, in the presence of cytokine cocktails [22
]. At least one report shows that suspension cultures of murine Lin- bone marrow culture can induce MN-RET after genotoxic drug treatment, but with a limited time window of approximately 2–3 days for generating RETs [25
]. This window does not adequately reflect in vivo conditions. Suspension cultures thus may not be most suitable for studying the kinetics of MN-RET induction.
LTBMC has been improved from the traditional Dexter’s bone marrow culture in flasks [26
]. These improved cultures support long-term growth and differentiation of erythroid progenitors and precursors [27
]. Wu and colleagues have further improved the performance by maintaining the 3D organization of bone marrow cells in the 3D bioreactor [19
], where the configuration mimics bone marrow structure in vivo and erythropoiesis is sustained up to several weeks [33
With improved 3D bioreactor and cytokine combinations, we have previously optimized a murine 3D LTBMC capable of generating high percentages of RETs for up to 8 weeks. We have demonstrated the system’s utility in a study of MN-RET induction following irradiation of mouse bone marrow [18
]. We observed parallel MN-RET induction in vivo after total body irradiation in C57Bl/6 mice when compared with MN-RET induction of bone marrow cultures of C57Bl/6 in 3D bioreactors with very similar radiation dose responses, as well as comparable patterns of kinetics of MN-RET induction after radiation [18
]. The investigation of human bone marrow in 3D LTBMC has required additional modifications of the culture condition, as the optimized culture condition for mouse bone marrow does not sustain the culture for human bone marrow.
Although enucleated erythrocytes have been observed in the human LTBMCs by other investigators, the percentage of RETs and the kinetics have not been reported [32
]. We have optimized serum, cytokines, and iron sources to establish a working system for generating RETs up to 4 weeks (), although full optimization may not have been achieved. The average RET percentage in the output from three cultures reached 11% on day 14 and was about 5% by day 28, suggesting that terminal erythroid differentiation was still active in the end of the culture. This condition has been tested in cultures from different healthy human donors, and appears robust despite the variation among different healthy donors. All cultures were able to generate sufficient RETs and radiation-induced MN-RET after irradiation with a wide range of doses from 1–6 Gy.
Gamma radiation caused robust MN-RET formation in the human 3D LTBMC. The kinetics of MN-RET induction from each individual were observed in our experimental design and shared common features. First, the background percentage of MN-RET post sham (0 Gy) radiation was consistently low and stable throughout the culture period. The percentage of MN-RET started to rise only “after” a latent period of approximately one-day post-radiation, with a significant increase by day 2. Maximum MN-RET induction was observed on day 3 after 1 Gy treatment in all cultures. With the exception of one donor, delayed peak to 3–4 days post-radiation was observed with radiation doses of 2 Gy, and to 4–5 days post-radiation with radiation doses ≥3 Gy (). A trend was evident whereby the peak of %MN-RET was delayed as radiation exposure levels increased in each culture. We would have preferred to include triplicate experimental data points for the presentations of and , but, given the limited yield of cells from each bone marrow donor and the need for multiple experimental data points of the 3D culture system, we were only able to include duplicate cultures. To minimize scoring biases, each data point was generated with the scoring of a minimum of 2000 RETs.
It is likely that higher doses of radiation led to longer cell-cycle delay [38
] and thereby to an increase of MN-RETs at later time points and, consequently, a wider peak as well. While our experimental design has covered radiation doses as high as 6 Gy, the peak value may not have reached the maximum for radiation doses beyond 6 Gy. There were fewer and fewer cells for scoring, as apoptotic effects dominated at higher radiation doses. We noted some variation of the peak time interval in higher dose radiation than in low dose radiation among different donors. For example, after 4 Gy exposure, the MN-RET percentage peaked on day 3 in three donor cultures, on day 4 in three other donor cultures, and on day 5 in another culture ( and ). This result contrasts with the peak MN-RET level on day 3 in all cultures after low dose radiation exposure (1 Gy). Apart from the potential cell-cycle delay, the underlying mechanisms of such variables can be confounded by differences in radiation sensitivity, DNA repair capacity, and nutrients involved in erythropoiesis among human population.
The increase of MN-RET from day 2 did not appear to be random, but instead was part of a pattern observed in most bone marrow samples we have examined. At least one cell division-cycle was required for the formation of micronuclei after radiation. The minimum time for the detection of MN-RET post radiation, i.e. the latent period, would have equaled the sum of time required by a cell division cycle and the enucleation event. While not much is known about the human MN-RET kinetics after radiation, MN-RET formation in mouse bone marrow and in the peripheral blood has been observed to peak at about 45 hours post-irradiation at 1 Gy [40
Our group has previously reported MN-RET induction in the human peripheral blood from cancer patients receiving partial body irradiation. We noted a latent period of 2–3 d in peripheral blood post radiotherapy in most cancer patients [6
]. We anticipate differences in the kinetics of human and mouse bone marrow. Likewise, we also expect differences in the kinetics of MN-RET in bone marrow and in the peripheral blood, as the spleen constantly removes MN-RET from the circulation. Others have estimated that a minimum of 2 d is needed for MN-RET to appear in the peripheral blood [41
]. Grawe et al. observed an approximate 3 d latent time from patients with normal erythropoiesis and argued that the shorter latent time in some patients was caused by iron-deficient erythropoiesis and the anemia suffered by them [12
Our data showed approximately 2–3 d latent time for MN-RET induction in the human 3D LTBMC after radiation, which suggested that the cell proliferative rate and cell cycle time of bone marrow progenitor and precursor cells for erythropoiesis are comparable between the human 3D LTBMC and human bone marrow in vivo [6
]. However, our data represents findings of the kinetics of MN-RET and RET production of bone marrow established in the 3D bioreactor, a synthetic bioculture system that sustains the generation of reticulocytes and erythrocytes from the upstream stem cells, progenitor cells, and precursor cells. The 3D bioculture system is not intended to simulate both the bone marrow and the peripheral blood compartments of humans, nor is it suitable to address the complexity of the interaction between the bone marrow and peripheral blood; the 3D culture system resembles only one compartment in its production of RETs and radiation-induced MN-RETs. It lacks the two-compartment steady-state of erythropoiesis of the in vivo condition, as there is constant relocation of RETs and MN-RETs to peripheral blood after a certain residence time in the bone marrow in vivo. To complicate the in vivo kinetics further, the spleen serves as a filter and constantly clears RETs and MN-RETs from the peripheral blood circulation. Thus the data generated from the 3D bioreactor at most resembles the condition in the bone marrow only and not the kinetics in the peripheral blood.
In summary, we report a 3D bone marrow culture system that has sustained long-term in vitro erythropoiesis. There is time and dose-dependent induction of MN-RET by gamma radiation in the human 3D LTBMC. A trend was evident whereby the peak of % MN-RET was delayed as radiation exposure levels increased in most donor cultures. Our data from human 3D LTBMC reveal novel findings of kinetics of radiation-induced MN-RET in a time and dose-dependent manner. Our data also support that the human 3D LTBMC model may offer great potential for investigating the induction of MN-RETs of human bone marrow by other genotoxic agents.