Precursor cells in the hematopoietic stromal microenvironment

Simultaneous i.v. injection of bone marrow cells to donors did not affect the size of the foci formed from either irradiated donors, or donors with previously curetted bone marrow; meaning that mesenchymal stem cell (MSCs) numbers in the femurs were not affected by the injected cells [14].

One of the most important factors providing for the formation of a full-grown hematopoietic microenvironment in vitro is the explantation of the BM as a whole, or in fragments, but not as a suspension [8]. When the BM is seeded as a suspension, cells do not form the complex stromal layer; on the contrary, cells form a thin monolayer with fibroblast colonies. No hematopoiesis is observed in such layers. Therefore the dissociation of bone marrow cells changes their differentiation potential. Stromal progenitors do not fulfill the variety of cellular differentiations leading to the formation of complex structures of a hematopoietic microenvironment in culture. Thus, the intercellular connections are of most importance for the preserving of the MSC’s features.

When explanted in fragments, bone marrow stromal cells, initially occupying a tiny part of the cultivation flask, built hematopoietic stromal structures on the whole flask surface. During this process they do not lose (or constantly renew) essential intercellular contacts. In order to find out whether this ability is present in the cells from a completely formed cell layer a “wound” was inflicted on the 3-week-old ACL. One week later fibroblastoid cells were observed only rarely on the wound site. No complex ACL was formed. Completely different results were obtained when the “wound” was inflicted on a 1-week-old cell layer, it being in the formation process. One week later the wound site was covered with typical for ACL stromal cells so tightly that it was hardly definable. The ACL regenerated not only morphologically but also functionally as was demonstrated by the ectopic foci formation (Table 2).

Table 2. Size of the foci formed by intact 3-week-old or regenerated ACL (ACL was wounded after 1 week of cultivation)

Thus, the intercellular contacts are of highest importance for the stromal progenitors’ differentiation during the processes of building the hematopoietic microenvironment. Stromal progenitor cells are able to keep the intercellular contacts while forming the microenvironment in vitro, and lose this ability when the functional adherent cell layer is formed [13].

The origin of stromal and hematopoietic cells in chimeras and the ectopic foci
Discriminant analysis showed that only MSCs of recipient origin are present in chimera bone marrow 6 and 12 months after irradiation and injection of hematopoietic cells (3–5x10⁶) (Table 3).

Table 3. Discriminant analysis of hemopoietic stromal progenitor origin in B6-in-CBF chimeras

Chimera marrow implanted to non-irradiated mice of the donor strain (B6) was always rejected, and the hematopoietic microenvironment could only be transferred to the recipient strain (CBF) [14]. This means, firstly, that stromal cells in the ectopic hematopoietic foci are of BM donor origin, and secondly, that BM stromal cells injected intravenously for the reconstitution of hematopoiesis after irradiation did not engraft bone marrow stroma of chimeras.

The hematopoietic cells in the ectopic foci were only of recipient origin, as was shown after implantation of adherent cells from B6 female bone marrow cultures under the renal capsule of B6 males (Table 4). The origin of hematopoietic cells in the focus was determined according to the presence or absence of a Y-chromosome [11]. 

Table 4. The origin of hematopoietic cells in ectopic hemopoietic foci produced by adherent cell layer from long-term bone marrow culture

The results show that stromal progenitors capable of hematopoietic microenvironment transfer cannot be transplanted i.v., and do not take part in MSC regeneration after irradiation. On implantation of BM of intact mice or adherent cell layer from bone marrow cultures under the renal capsule, ectopic hematopoietic foci form, in which the hematopoietic cells belong to the recipient while the stroma is of donor origin.

In order to investigate the origin of stromal cells in ACL, LTBMCs had been established from B6-in-CBF1chimeras, and when stable ACLs had been formed they were carefully scraped as a whole and implanted under the renal capsule in both B6 and CBF1 recipients. This discriminant analysis showed that ACL implantation produced ectopic foci only in the recipient line (CBF1) (Table 5).

Table 5. Discriminant analysis of hematopoietic stromal progenitors originating in adherent cell layer (ACL) of long-term bone marrow cultures of B6-in-CBF1 chimeras

Thus, MSCs in the ACLs from long-term bone marrow cultures of chimeras are only of recipient origin [14].

Linear interdependency between the foci size and amount of MSCs implanted

Various amounts of medullary tissue ranging from 1/4 to 4 femoral bone marrow plugs were transplanted and the hematopoietic cells were counted in the foci formed 1 month later. The results are shown in Table 6, where it can be seen that despite the small number of implants (3–7), the number of nucleated cells on the whole showed a linear relation with the size of the implanted bone marrow fragment. The correlation coefficient (r) was 0.97±0.014 and the extrapolation number 4.68x10^-5 [5].

Table 6. The influence of ectopic marrow implant size on hematopoietic cell number in the foci formed

In summary, stromal cells implanted under the renal capsule of syngeneic animal form a hematopoietic microenvironment only if MSCs are preserved among them, and the size of these hematopoietic foci depends solely on the number of MSC among implanted stromal cells. These results allow MSC to be studied on this model.

The size of ectopic foci is proportional to the amount of ACL implanted. ACL taken from the half of the flask bottom produces a focus that is approximately half that formed by ACL collected from the whole surface of the culture flask: 35.6x10⁶ and 58.1x10⁶ nucleated cells, respectively (correlation coefficient is 0.996±0.005) [12].

When transplanted under the renal capsule of a syngeneic recipient, ACL of cultures of a single femur creates a focus approximately the size of a focus formed in implantation of bone marrow freshly isolated from a single femur. This coincidence suggests that the content of stroma precursors in the culture corresponds to the explanted bone marrow dose but not to other factors, for instance, the surface of the flask bottom. In view of this, the size of foci produced by ACL from 4–6-week-old cultures of 1/2, 1 and 2 femurs was studied (Table 7).

Table 7. Correlation between bone marrow dose plated in LTBMC and the size of the ectopic foci formed

The size of the foci was linearly associated with the dose of the implanted bone marrow (correlation coefficient 0.999±0.001) [12].

Kinetics of stromal precursor cell proliferation in vitro and during ectopic foci formation
The time-course of the stromal precursor repopulation during the process of a creating a site of ectopic hematopoiesis was also studied. In the first 6 hrs after implantation the number of transplantable stromal precursors reduced appreciably, reaching the nadir by the end of the first day (about 20% of the number implanted). Thereafter, there was a phase of regeneration and stromal precursors recovered up to the initial level in 3 weeks. The sensitivity of the stromal precursors to the cytostatics is in good agreement with such kinetics. Normally the stromal precursors do not actually show proliferative activity, which is seen from their insensitivity to MTX. Twenty-four hours after implantation their proliferative activity remains low. During the next 24 hrs the precursors are triggered into the cell cycle synchronously. At this time up to two-thirds of the stromal precursors are killed by the cytostatics. High sensitivity to the cytostatics is observed at 3–4 and 9–10 days after implantation, but not 5–6 days after. It is not clear whether these fluctuations are incidental, or if they are related to the movement of the partially synchronized cell population through the cell cycle. Three weeks later, i.e., when the number of stromal precursors had recovered, their proliferative activity decreased to the initial low level, and remained on that level [15].

Stromal precursors also proliferate during the formation of the adherent cell layer in the LTBMC. Considering the fact that hydroxyurea affects precursors from day 2 until day 11, it seems that these cells change their proliferative status slowly, for more than 24 hours. Thus, both the implantation of BM in vivo, leading to the building of a new hematopoietic microenvironment and hematopoietic foci formation, and the explantation of BM in vitro, leading to the building of a hematopoietic microenvironment in the form of an adherent cell layer in LTBMC, are accompanied by identical changes in the proliferative status of stromal precursors. In the first 24 hours after the transfer they remain at mitotic rest, then, during the next 2 days they mobilize into the cell cycle substantially and synchronically. For the next 2 weeks they are highly active in proliferation. Afterwards, despite continuous growth of ectopic foci or ACLs, the stromal precursors do not proliferate. This is reflected in the absence of increase in the number of stromal precursors both in vivo and in vitro after 3 weeks of formation of the microenvironment.

Characteristics of stromal progenitors in vivo and in vitro

De novo formation of a stromal microenvironment after the implantation of MSC under the renal capsule
In bone marrow implantation the hematopoietic cells leave the graft, whereas the stromal precursors form a new hematopoietic stroma, which is then repopulated by host cells. The cellularity of the hematopoietic focus is proportional to the initial implant size, i.e., to the content of the stromal precursors in it. On retransplantation of the intact ectopic site, hemopoietic cells again leave the implant, as they do after the primary implantation of the bone marrow plug. Twenty-four hours after retransplantation no more than about 3% of the CFU-S remain in the focus (Table 8) [15].

Table 8. Cellularity and CFU-S content of an ectopic site of hematopoiesis as a function of time after retransplantation

The replacement of the hematopoietic cells by the recipient cells in the retransplanted focus was also confirmed karyologically [16]. Hence, it appears that when the formed site of ectopic hematopoiesis is retransplanted, the hematopoietic microenvironment is created de novo.

Self-renewal ability of MSC
The results permitted the study of the capacity of MSCs for repeated formation of ectopic foci. Nine passages failed to produce any reduction in size of the newly formed hematopoietic foci (Table 9).

Table 9. Cellularity of ectopic bone marrow foci on repeated transplantation

During the serial transfer of the ectopic hematopoietic tissue without ossicles, a complete loss of the ability to form the hematopoietic focus was already apparent at the third passage. In this case no more than half of the stromal precursors remained on the ossicle (when the bone marrow is pressed out of the femur only 10–15% of the stroma precursors remain on the bone), which was verified by separate implantation of the ossicle and hematopoietic tissue from focus [15].

The self-maintenance ability of the stromal precursors was also studied in a model in which the medullary cavity was repeatedly curetted. Four successive curettages were carried out, and in each over 90% of the stromal precursors were washed out of the femur. After each curettage, the complement of stromal precursors recovered over 1–1.5 months up to 50–60% of the initial and after the fourth curettage up to 20%. Subsequent curettages proved impossible because the whole medullary cavity was filled with newly formed bone [15].

The capacity for self-maintenance of stromal precursors from cultures was studied by repeated transfer of ectopic hemopoietic foci created by them in intermediate to final recipients (Table 10).

Table 10. Self-maintenance ability of hematopoietic stromal precursors from long-term bone marrow cultures

The self-maintenance of MSCs from LTBMC proved to be low and the size of the secondary foci was 10–15% of that of foci in the intermediate recipients [11]. Thus, MSCs are maintained in LTBMC and are capable of creating a hematopoietic microenvironment on transplantation. However, self-maintenance of these precursors from LTBMC is essentially diminished.

The data obtained have demonstrated a high ability of self-maintenance of the cells transferring the hematopoietic microenvironment. Twenty-four hours after BM implantation about 10% of the stromal precursors survive. The population of the precursors in the femur is reduced to approximately the same degree after bone marrow curettage. During regeneration the stromal precursors increase to 60–100% of the initial level, hence they must undergo three or four mitoses. This is consistent with the rise in their sensitivity to S-phase specific cytostatics for the first 2 weeks after implantation. Taking into consideration that on the serial transfer of hematopoietic tissue without ossicles or on serial curettage three or four cycles of regeneration are possible, the stromal precursors are able to undergo no less than 10–12 mitoses. This value is rather underestimated since calculating the loss of precursors for differentiation was not taken into account. The high self-maintenance ability, on the one hand, and the ability to form a fully differentiated bone marrow stromal tissue on the other, suggests that the cells transferring the hematopoietic microenvironment are true mesenchymal stem cells.

Radiosensitivity of MSCs
The hematopoietic stroma function of the femoral bone marrow exposed in vitro to 500 to 2700 rad of γ-rays was also studied, using the ectopic foci formation method. Irradiation of bones with 500 rad caused no noticeable damage to the MSCs’ ability to form a hematopoietic microenvironment. Higher doses produced an exponential decrease in MSCs. The D₀ estimated from linear regression (regression equation: log survival=-0.000977x+0.7200) of this portion of the curve is 444±5 rad and the extrapolation number (n) is 5.2. The results for in vitro neutron irradiation of bone marrow agreed (regression equation: log survival=-0.002697x+0.1506). A small shoulder was evident followed by an exponential survival having D₀ and n of 161±19 rad and 1.4, respectively [5].

The radiosensitivity of MSCs from 5-week-old LTBMC seemed very similar to that of non-cultivated ones (regression equation: log survival=-0.089x+0.3744; D₀ was 486±15 rad and n was 2.4) [17].

When high doses of irradiation were used, implantation proved unsuccessful in some cases and no ossicles with hematopoiesis were formed. One may assume that not a single MSC is preserved in such implants. The independent and random character of radiation damage of cells suggests that the existence or nonexistence of MSCs in the implant is governed by Poisson’s distribution. In this case the data on the proportion of transplant failures (P₀) allow the mean MSC content in the implant (x) to be calculated using the equation x=- ln P₀. The total MSC content in the femoral bone marrow can be found by taking into account the fraction that survived exposure to the given dose. These data are shown in Table 11 [5] where it is seen that the results were quite consistent on the whole.

Table 11. The effect of g irradiation on the hematopoietic microenvironment transferring MSCs in murine femoral bone marrow

The mean number of MSCs calculated from the proportion of transplant failures after high doses of irradiation was 52.3±11.6 per femoral bone marrow plug.

The results show that MSCs are much more radioresistant than hematopoietic stem cells, for which D0 is about 100 rad. The second feature of MSCs is their marked capability to recover from sublethal γ-ray damage, which is characterized by a extrapolation number (5.2). In the case of neutron irradiation the extrapolation number is 1.4, which is evidence that MSCs are incapable of recovery from sublethal neutron-induced damage [5].

Despite their high radioresistance MSCs were still sensitive to irradiation. After lethal irradiation and syngeneic bone marrow transplantation of CBF1 mice, the MSCs were reduced to 1/5 of the initial level and slowly regenerated to subnormal level in 6 months. The number of bone marrow cells used for reconstitution of the primary recipient did not affect MSC regeneration (Table 12) [14].

Table 12. Hematopoietic stromal precursors in lethally irradiated CBF1 mice reconstituted with different doses of syngeneic bone marrow cells

Two to six months after irradiation, the content of stromal progenitors in the mouse femur was, judging from the cellularity of the foci produced by them, 40–50% of the initial level in both groups of mice reconstituted both in minimal protective dose of marrow cells (2x10⁵) and with a hundredfold dose. Thus, MSCs could be affected by high doses of irradiation and in such cases they are not able to regenerate completely.

Influence of the quality of hematopoiesis on MSCs’ proliferative potential
MSCs were compared in chimeras and double chimeras. Hematopoietic foci formed in standard recipients via BM from chimeras were approximately 2 times smaller when compared with foci formed from BM of non-irradiated mice, and 2 times bigger compared to foci formed from BM from double chimeras. These data confirm that stromal precursors are radiosensitive and unable to recover from radiation damage completely. Interestingly, secondary and tertiary chimeras’ BM formed the same small ectopic foci as double chimeras did, while the dose of irradiation affected the stroma of secondary and tertiary chimeras was 2 times lower than of double chimeras [18]. When BM from all types of chimeras tested were explanted in LTBMC, and after 3–4 weeks of cultivation were implanted under the renal capsule of syngeneic mice, foci formed from ACLs from chimeras’ BM were 75%, foci formed from ACLs from double chimeras’ BM were 20%, and ACLs from secondary and tertiary chimeras were approximately 40% by size from ACLs from non-irradiated mice. The secondary and tertiary chimeras’ stromal precursors were irradiated only once while hematopoietic cells had undergone 2 or 3 rounds of intensive proliferation during reconstitution of hematopoiesis. Thus, judging by the grade of irradiation damage, stromal cells from secondary and tertiary chimeras were similar to ordinary chimeras. Nevertheless, secondary and tertiary chimeras’ stromal precursors turned out to be affected more deeply than the same cells in ordinary chimeras. The data indicates that the stroma’s damage was determined not only by the irradiation dosage but by the quality of hematopoietic cells proliferating on it [18].

Hierarchical organization of the MSC compartment: existence of more mature than MSC-inducible precursor cells

In BM implantation, the ectopic hemopoietic focus is larger in an irradiated recipient than in a non-irradiated one in a dose-dependent manner (Table 13) [19].

Table 13. The size of the ectopic foci in irradiated recipients

In subsequent passages the size of the focus did not increase further (Table 14, compare with Table 9).

Table 14. Cellularity of ectopic bone marrow foci on repeated transplantation into irradiated recipients

Hence, one may conclude that in the irradiated recipient the number of stromal precursors in an ectopic focus does not correspond to the large size of the focus. This was demonstrated more directly by implantation of similar bone marrow fragments into non-irradiated and irradiated (chimeric) recipients, subsequently testing the content of the stromal precursors in the sites formed by their retransplantation to non-irradiated recipients. In these experiments the primary focus formed in chimeras exceeded that in the non-irradiated ones by a factor of 2.2 (27.3x10⁶ and 12.5x10⁶). The content of the stromal precursors in both was essentially the same since the cellularities of the foci formed on transfer to the non-irradiated recipients were 10.93x10⁶ and 11.83x10⁶, respectively [15]. Stromal precursors from a culture also react to stimulation from the irradiated recipient, the response being much stronger than in implantation of freshly isolated bone marrow: the foci were 5–7 times larger in the irradiated recipients than in the non-irradiated ones (35.1±8x10⁶ versus 5.3±1.2x10⁶) (Table 15) [11].

Table 15. Size of ectopic foci produced by adherent cell layer from LTBMC in non-irradiated and irradiated recipients

Similar results were obtained when LTBMC were established from different amounts of bone marrow (1/2, 1, 2 femurs). All cultures proved to be alike in hematopoiesis maintenance, though the MSC content in them corresponded to the dose of plated bone marrow (see Table 7). At the same time, in irradiated recipients all cultures produced foci approximately the same size (37.1, 42.7 and 38.3x10⁶ correspondingly) [12].

The effect of hydroxyurea was estimated by the size of ectopic foci formed from treated ACLs in irradiated and non-irradiated recipients [20]. The results were similar in both types of recipients. So one may conclude that during the ACL formation both types of stromal precursors actively proliferate. MSCs functioning during the transfer into non-irradiated recipients and the more mature precursors taking part in the foci formation in the irradiated recipients.

The data suggest that the compartment of the hemopoietic stromal precursors is heterogenic and includes cells of at least two differentiation levels. Those less differentiated, MSCs able to transfer a hematopoietic microenvironment are marked by a relatively high self-maintenance, do not respond to the systemic demand of the irradiated recipient, and create a microenvironment, the size of which is proportional to the number of transferred MSCs. More mature precursors are marked by poor self-maintenance; they respond to the systemic demand of the irradiated recipient and, in the culture, to the surface of the flask bottom, and are not transplantable via in vivo transfer.

Summarizing the data presented above, the compartment of stromal bone marrow cells has a hierarchical structure. There are true mesenchymal stem cells (MSCs) capable of both self-maintaining and differentiating into all stromal lineages, and more mature inducible stromal precursors, which keep the ability for multipotential differentiations while losing their self-renewing ability. MSCs are mainly in mitotic rest and are not sensitive to the systemic demand, while more mature precursors proliferate more easily in the case of systemic requirements.

More mature inducible stromal precursors are sensitive to the unknown factor(s) released after irradiation. When injected during foci formation sera from irradiated mice also stimulate these precursors, resulting in increased size of the foci formed – the size of the foci was 19.5±1. 8x10⁶ compared with 14±1.5x10⁶ in control mice [19]. Addition of sera from irradiated mice to the LTBMC also increases the number of stromal cells in the ACLs (Table 16) [21].

Table 16. Influence of the sera from irradiated mice on the number of cells in the ACLs from LTMBC

In order to define the organ producing the unknown factor(s), various organs of irradiated mice were co-transplanted simultaneously with the implantation of the BM plug under the renal capsule. Intravenous injection of irradiated sera as well as implantation of 5–6 irradiated bones under the skin enhanced the growth of stromal cells in the foci formed (Table 17) [21].

Table 17. Analysis of the various organs of irradiated mice for their stroma-stimulating activity

Thus, the unknown factor(s) stimulating the growth of stromal inducible precursor cells are produced in the irradiated bones and secreted into the blood serum.

The regulation of MSCs in vivo by hematopoietic growth factors
Regulation of MSCs by soluble factors remains obscure. During the formation of the hematopoietic microenvironment MSCs are affected by G-CSF. When recipients of BM were injected over 10 or 17 days beginning from the day after implantation of BM under the renal capsule it dramatically decreased size of the foci formed (Table 18).

Table 18. Influence of G-CSF on the ectopic foci formation from the bone marrow of intact mice

Obviously, when administered during the active proliferation and differentiation of MSC, G-SCF inhibits their proliferation (judging from the decreased size of the foci formed); moreover, it diminishes the number of MSCs themselves as was shown by retransplantation of the foci [22].

Conversely, cytokine treatment of the MSCs in their steady-state (in the intact bone marrow increases the number of these stromal precursors (Table 19).

Table 19. Size of the ectopic foci formed from the bone marrow of mice treated with cytocines

The combination of G-CSF with SCF and the longest treatment (for 17 days) had a maximal effect on the MSC number in the bone marrow of treated mice. In the case of G-CSF treatment this effect was transient and did not last for a month, in the case of cytokine combination, however, the increase in the number of MSCs was stable for at least a month. Cytokine treatment did not affect the osteogenic potential of MSCs either during stroma formation or in the intact bone marrow [22].

Thus, during the building of ectopic hematopoietic foci when stromal precursors are proliferating and differentiating, pharmacological concentrations of G-CSF inhibit the process of foci formation and diminish the number of stromal precursors in them. When affecting the mature non-proliferating bone marrow stroma, cytokines increase the number of MSCs.

Conclusions

Summarizing the works of J. L. Chertkov, it is possible to describe the main features of MSCs. These cells are capable of transferring a hematopoietic microenvironment due to both their high proliferative potential and their ability to differentiate into all bone marrow stromal lineages, including bone, cartilage, and marrow stromal cells. MSCs are more radioresistant than HSCs but they still suffer from radiation and their damage could not be fully recovered. Impaired hematopoiesis also influences the MSCs. The compartment of MSCs and stromal precursor cells is organized hierarchically. Therefore, J. L. Chertkov described the main features of MSCs from many sides using the functional assay.

Acknowledgements

Irina Shipounova and Nina Drize for preparing the compilation of Chertkov’s works on hematopoietic stromal microenvironment.

Gurevich O. A., Udalov G. A., Samoylina N. L., Samoylova R. S., Lemeneva N. L., Todria T. V., Olovnikova N. I., Olshanskaya Y. V., Shiponova (Nifontova) I. N., Ershler M. A. and Drize N. I. as co-authors of his works.

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