Alleviation of acute radiation-induced bone marrow failure in mice with human fetal placental stromal cell therapy

Abstract

Purpose: Selected placental mesenchymal stromal cells isolated from the fetal mesenchymal placental tissues (f-hPSCs) were tested as cell therapy of lethal acute radiation syndrome (ARS) with bone marrow regeneration and induced extramedullary hematopoiesis.

Methods and materials: f-hPSCs were isolated from the chorionic plate of human placentae and further expanded in regular culture conditions. 2 × 10⁶ f-hPSCs were injected on days 1 and 4 to 8-Gy total body irradiated (TBI) C3H mice, both intramuscularly and subcutaneously. Pre-splenectomized TBI mice were used to test the involvement of extramedullary spleen hematopoiesis in the f-hPSC-induced hematopoiesis recovery in the TBI mice. Weight and survival of the mice were followed up within the morbid period of up to 23 days following irradiation. The role of hematopoietic progenitors in the recovery of treated mice was evaluated by flow cytometry, blood cell counts, and assay of possibly relevant growth factors.

Results and conclusions: The survival rate of all groups of TBI f-hPSC-treated mice at the end of the follow-up was dramatically elevated from < 10% in untreated to ~ 80%, with a parallel regain of body weight, bone marrow (BM) recovery, and elevated circulating progenitors of blood cell lineages. Blood erythropoietin levels were elevated in all f-hPSC-treated mice. Extramedullary splenic hematopoiesis was recorded in the f-hPSC-treated mice, though splenectomized mice still had similar survival rate. Our findings suggest that the indirect f-hPSC life-saving therapy of ARS may also be applied for treating other conditions with a failure of the hematopoietic system and severe pancytopenia.

Keywords: Acute radiation syndrome (ARS); Bone marrow; C3H mice; Extra-medullary hematopoiesis (EMH); Fetal human placental stromal cells (f-hPSCs); Hematopoiesis; Hematopoietic stem cells (HSC); Spleen.

Fig. 1

Experimental set-up and follow up of mice weight and survival. a Experimental set up. TBI of 8 Gy was done on day 0. The 2 × 10⁶ f-hPSCs were injected IM (IM-f-hPSCs) or SC (SC-f-hPSCs) on days 1 and 4. Pre-splenectomized mice [Spl-] were treated only with IM f-hPSC injections. Weight and survival were followed up for up to 23 days (b, c, respectively). Non-irradiated f-hPSC-treated and non-treated Naïve mice served as controls

Fig. 2

The CBC profile of the survivors at the termination of the follow-up. WBC, RBC, and PLT counts and RDW were measured at the end of the follow-up for all the experimental groups tested. a–d Giemsa stained blood smears detect prematurely released reticulocytes to the circulation (e) (p value: *< 0.05, **< 0.01, ***< 0.001, ****< 10⁻³)

Fig. 3

Histological sections of the bones and BM cellularity of the different groups tested. Representative cross-sections of tibia of mice at the termination of the follow-up stained with H&E to observe the degree of regeneration of the BM (a). A typical area in each bone section is further magnified. The difference in the cellularity of the BM in each group is demonstrated. Total BM cell counts obtained from the 4 long bones of all mice, frequency of HSCs, and total numbers of HSCs in the different groups are presented in (b–d), respectively

Fig. 4

Spleen and liver sites of extramedullary hematopoiesis. a Spleen weight at the termination of the follow-up. Increased spleen weight was associated with elevated number of megakaryocytes only in TBI IM or SC f-hPSC-treated mice, as compared to the naïve controls (b). The spleen cellularity and size, which may represent the extent of spleen damage which varied in the few surviving Veh-Cont mice, are presented (c Vs d, with magnification of selected area). Increased total number of spleen cells and the presence of HSCs (identified as Lin- negative, [cKit+, SCA1+ and CD150+) in f-hPSC-treated mice, as compared with Veh-Cont and Naïve mice, may suggest increased Spleen-EMH. Total numbers of isolated cells, HSC frequency, and their total counts in the spleen are presented in e–g (p values:*< 0.05, **< 0.01, ***< 0.001, ****< 0.0001)

Fig. 5

Maturation state of erythropoietic cells in the spleen and in the BM of the groups tested. a Flow cytometry of the different stages of erythropoiesis in the BM and spleen in mouse groups as tested with cell-markers CD71 and Ter119 [27]. EryA cells are less mature, large proliferating erythropoietic progenitors, both CD71⁺ and TER119⁺. EryB cells are smaller CD71⁺ cells. EryC representative is most mature small non dividing erythrocytes. Distribution of these cell populations harvested from the BM and spleen at the termination of the follow-up is presented in b. A comparison of Ter119 Cy3 nucleated cells staining of liver cryosections of non-operated and |Spl-] f-hPSC-treated TBI mice (c). Only a few individual cells (marked by a circle) where stained in the liver sinusoids, with no apparent liver extramedullary erythropoiesis, in spite of the higher hematopoietic stress in [Spl-] group due to the absence of spleen-EMH (p values: *> 0.05, **> 0.01, ***> 0.005)

Fig. 6

Plasma EPO and m-VEGF levels on day 23, at the termination of the follow-up. On day 7 of the follow-up, concentrations of human and murine EPO (a) and VEGF (b) were measured in the blood plasma of the TBI mice, treated or untreated with f-hPSCs. EPO level was significantly elevated in TBI mice and further increased in f-hPSC-treated mice. h-VEGF was not detected, but m-VEGF reached detectable levels and was significantly higher in the untreated TBI mice, relative to the Naïve or f-hPSC-treated TBI mice (p value: *< 0.05, **< 0.01)