Improving hematopoietic recovery through modeling and modulation of the mesenchymal stromal cell secretome.

BACKGROUND: Efficient and sustained hematopoietic recovery after hematopoietic stem cell or bone marrow transplantation is supported by paracrine signaling from specific subpopulations of mesenchymal stromal cells (MSCs). Here, we considered whether in vitro mechanopriming of human MSCs could be administered to predictively and significantly improve in vivo hematopoietic recovery after irradiation injury. METHODS: First, we implemented regression modeling to identify eight MSC-secreted proteins that correlated strongly with improved rescue from radiation damage, including hematopoietic recovery, in a murine model of hematopoietic failure. Using these partial least squares regression (PLSR) model parameters, we then predicted recovery potential of MSC populations that were culture expanded on substrata of varying mechanical stiffness. Lastly, we experimentally validated these predictions using an in vitro co-culture model of hematopoiesis and using new in vivo experiments for the same irradiation injury model used to generate survival predictions. RESULTS: MSCs grown on the least stiff (elastic moduli ~ 1 kPa) of these polydimethylsiloxane (PDMS) substrata secreted high concentrations of key proteins identified in regression modeling, at concentrations comparable to those secreted by minor subpopulations of MSCs shown previously to be effective in supporting such radiation rescue. We confirmed that these MSCs expanded on PDMS could promote hematopoiesis in an in vitro co-culture model with hematopoietic stem and progenitor cells (HSPCs). Further, MSCs cultured on PDMS of highest stiffness (elastic moduli ~ 100 kPa) promoted expression of CD123+ HSPCs, indicative of myeloid differentiation. Systemic administration of mechanoprimed MSCs resulted in improved mouse survival and weight recovery after bone marrow ablation, as compared with both standard MSC expansion on stiffer materials and with biophysically sorted MSC subpopulations. Additionally, we observed recovery of white blood cells, platelets, and red blood cells, indicative of complete recovery of all hematopoietic lineages. CONCLUSIONS: These results demonstrate that computational techniques to identify MSC biomarkers can be leveraged to predict and engineer therapeutically effective MSC phenotypes defined by mechanoprimed secreted factors, for translational applications including hematopoietic recovery.

Cytogenetic Reconstruction of Gamma-Ray Doses Delivered to Atomic Bomb Survivors: Dealing with Wide Distributions of Photon Energies and Contributions from Hematopoietic Stem/Progenitor Cells.

Retrospective estimation of the doses received by atomic bomb (A-bomb) survivors by cytogenetic methods has been hindered by two factors: One is that the photon energies released from the bomb were widely distributed, and since the aberration yield varies depending on the energy, the use of monoenergetic 60Co gamma radiation to construct a calibration curve may bias the estimate. The second problem is the increasing proportion of newly formed lymphocytes entering into the lymphocyte pool with increasing time intervals since the exposures. These new cells are derived from irradiated precursor/stem cells whose radiosensitivity may differ from that of blood lymphocytes. To overcome these problems, radiation doses to tooth enamel were estimated using the electron spin resonance (ESR; or EPR, electron paramagnetic resonance) method and compared with the cytogenetically estimated doses from the same survivors. The ESR method is only weakly dependent on the photon energy and independent of the years elapsed since an exposure. Both ESR and cytogenetic doses were estimated from 107 survivors. The latter estimates were made by assuming that although a part of the cells examined could be lymphoid stem or precursor cells at the time of exposure, all the cells had the same radiosensitivity as blood lymphocytes, and that the A-bomb gamma-ray spectrum was the same as that of the 60Co gamma rays. Subsequently, ESR and cytogenetic endpoints were used to estimate the kerma doses using individual DS02R1 information on shielding conditions. The results showed that the two sets of kerma doses were in close agreement, indicating that perhaps no correction is needed in estimating atomic bomb gamma-ray doses from the cytogenetically estimated 60Co gamma-ray equivalent doses. The present results will make it possible to directly compare cytogenetic doses with the physically estimated doses of the survivors, which would pave the way for testing whether or not there are any systematic trends or factors affecting physically estimated doses.

Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency.

During B and T lymphocyte maturation, V(D)J recombination is initiated by creation of DNA double-strand breaks. Artemis is an exonuclease essential for their subsequent repair by nonhomologous end-joining. Mutations in DCLRE1C, the gene encoding Artemis, cause T-B-NK+ severe combined immunodeficiency (ART-SCID) and also confer heightened sensitivity to ionizing radiation and alkylating chemotherapy. Although allogeneic hematopoietic cell transplantation can treat ART-SCID, conditioning regimens are poorly tolerated, leading to early mortality and/or late complications, including short stature, endocrinopathies, and dental aplasia. However, without alkylating chemotherapy as preconditioning, patients usually have graft rejection or limited T cell and no B cell recovery. Thus, addition of normal DCLRE1C cDNA to autologous hematopoietic stem cells is an attractive strategy to treat ART-SCID. We designed a self-inactivating lentivirus vector containing human Artemis cDNA under transcriptional regulation of the human endogenous Artemis promoter (AProArt). Fibroblasts from ART-SCID patients transduced with AProArt lentivirus showed correction of radiosensitivity. Mobilized peripheral blood CD34+ cells from an ART-SCID patient as well as hematopoietic stem cells from Artemis-deficient mice demonstrated restored T and B cell development following AProArt transduction. Murine hematopoietic cells transduced with AProArt exhibited no increase in replating potential in an in vitro immortalization assay, and analysis of AProArt lentivirus insertions showed no predilection for sites that could activate oncogenes. These efficacy and safety findings support institution of a clinical trial of gene addition therapy for ART-SCID.

Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM.

Cellulose ester membranes (CEM) were coated with stromal cells from murine bone or bone marrow irradiated in vitro with 1000, 2000, or 4000 rad and then implanted i.p. in CAF1 mice for periods of six and 12 months. CEM coated with stromal cells from bone showed excellent regeneration of bone and hematopoiesis after 1000 rad in vitro irradiation. After 2000 rad, hematopoietic and bone regeneration was reduced by about 50%, and after 4000 rad it was completely absent in CEM coated with stromal cells from bone. CEM coated with stromal cells from bone marrow showed no regeneration of hematopoiesis or bone after 1000, 2000, and 4000 rad in vitro irradiation and residence i.p. for six and 12 months. These results indicate that regeneration of the hematopoietic microenvironment is dependent upon living stromal cells. A difference in radiation sensitivity is demonstrated between stromal cells from bone and from bone marrow.

Restoration of femoral GM-CFC progenitors in sublethally irradiated mice of various ages treated with liposomal adamantylamide dipeptide.

In this study we tested the stimulatory effect of adamantylamide-l-alanyl-d-isoglutamine (AdDP) or its liposomal formulation (L-AdDP) on recovery of the granulocyte-macrophage hemopoietic progenitor cells in the bone marrow of sublethally irradiated mice of various ages. Number of GM-CFC progenitors in femur on day 10 was used as a parameter reflecting the stimulatory activity. Mice (aged 3-5 month) pre-treated with AdDP or L-AdDP via s.c. route displayed enhanced recovery of the granulocyte-macrophage hemopoietic progenitor cells at the dose of 5.5 Gy. Overaged mice (2 years) responded to the treatment when the dose was increased to 6.5 Gy, while radiation doses below 5.5 Gy should be used to see the stimulation effect in young mice (6 weeks). Entrapment of AdDP into liposomes enhanced costimulatory activity of sera of treated mice and prolongated this activity at least for 30 h after stimulation, in comparison to the mice treated with free AdDP where the costimulatory activity was spanned only up to 12 h. In conclusion, L-AdDP represents a suitable formulation of AdDP that induced recovery of GM-CFC progenitors in the femur of irradiated mice of various ages. The stimulatory effect depends on the extent of injury to bone marrow hemopoietic microenvironments caused by various doses of gamma-irradiation.

Liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE) promotes haemopoietic recovery in irradiated mouse.

Pretreatment of C57B1/6 mouse with the macrophage activator muramyl tripeptide phosphatidylethanolamine encapsulated in liposomes (MTP-PE/MLV) induced haemopoietic recovery in subsequently irradiated mouse. An optimal endoCFU-S survival was observed when 200 micrograms MTP-PE/MLV was administered i.p. 24 h before irradiation. MTP-PE/MLV did not affect the day 8 exogenous CFU-S survival in the bone marrow immediately after irradiation. However, 3, 6, 9 and 14 days after irradiation the number of day 8 CFU-S was almost 2 to 4-fold higher in the bone marrow of the MTP-PE/MLV injected mouse. Also, recovery of the GM-CFC pools in femoral bone marrow after irradiation proceeded at a faster rate in the MTP-PE/MLV-treated animal than in control groups. After a single i.p. injection of MTP-PE/MLV to the non-irradiated mouse, the number of CFU-S in bone marrow was not significantly different from controls, whereas the number of GM-CSC was significantly increased. In addition, the percentage of day 8 CFU-S and GM-CFC in S-phase of the cell cycle was significantly increased, as was colony-stimulating activity present in the serum of treated animals. Pretreatment with MTP-PE/MLV protected the C57Bl/6 mouse in a dose-dependent manner from the lethal effects of ionizing radiation. A single dose (100 or 200 micrograms) injected i.p. 24 h, or 100 micrograms MTP-PE/MLV injected i.v. 24 h before 9.5 Gy gamma-rays protected 47, 85 and 59% of C57B1/6 mouse, respectively. The dose reduction factor in the case when the MTP-PE/MLV (200 micrograms per mouse) was administered i.p. at that time was 1.17 (95% CL 1.13, 1.21). Combined administration of MTP-PE/MLV (24 h) and indomethacin (24 and 3 h) to mouse prior to irradiation exerted an additional radioprotective effect.

Effect of liposomal muramyl tripeptide phosphatidylethanolamine and indomethacin on hematopoietic recovery in irradiated mice.

The effects of liposomal muramyl tripeptide phosphatidylethanolamine (MTP-PE/MLV, radioprotective immunomodulator; 10 mg/kg) and indomethacin (INDO, inhibitor of prostaglandin production; 2 mg/kg) on post-irradiation recovery of hematopoietic functions in mice were investigated. Two agents with distinct radioprotective mechanisms were administered alone or in combination 24 h and 3 h before exposure to 7 Gy (60)Co radiation. In the post-irradiation period (3-14 days) combined pre-treatment of mice accelerated recovery of bone marrow cellularity, weight of spleen and myelopoietic and erythropoietic activity in both hematopoietic organs, compared to treatment with MTP-PE/MLV or indomethacin alone. In the peripheral blood, improved radioprotective effects of combined drug administration were found in the recovery of reticulocytes and platelet count. No further significant differences in the recovery of leukocyte count were observed in the examined groups until post-irradiation day 14. Within the first 3-6 post-irradiation days, the bone marrow and peripheral blood smears of mice pre-treated with indomethacin alone or its combination with MTP-PE/MLV more frequently featured blast cells and large cells with abundant cytoplasm which could be considered the hematopoietic stem cells.

Amelioration of radiation-induced oral cavity mucositis and distant bone marrow suppression in fanconi anemia Fancd2-/- (FVB/N) mice by intraoral GS-nitroxide JP4-039.

The altered DNA damage response pathway in patients with Fanconi anemia (FA) may increase the toxicity of clinical radiotherapy. We quantitated oral cavity mucositis in irradiated Fanconi anemia Fancd2(-/-) mice, comparing this to Fancd2(+/-) and Fancd2(+/+) mice, and we measured distant bone marrow suppression and quantitated the effect of the intraoral radioprotector GS-nitroxide, JP4-039 in F15 emulsion. We found that FA mice were more susceptible to radiation injury and that protection from radiation injury by JP4-039/F15 was observed at all radiation doses. Adult 10-12-week-old mice, of FVB/N background Fancd2(-/-), Fancd2(+/-) and Fancd2(+/+) were head and neck irradiated with 24, 26, 28 or 30 Gy (large fraction sizes typical of stereotactic radiosurgery treatments) and subgroups received intraoral JP4-039 (0.4 mg/mouse in 100 µL F15 liposome emulsion) preirradiation. On day 2 or 5 postirradiation, mice were sacrificed, tongue tissue and femur marrow were excised for quantitation of radiation-induced stress response, inflammatory and antioxidant gene transcripts, histopathology and assay for femur marrow colony-forming hematopoietic progenitor cells. Fancd2(-/-) mice had a significantly higher percentage of oral mucosal ulceration at day 5 after 26 Gy irradiation (59.4 ± 8.2%) compared to control Fancd2(+/+) mice (21.7 ± 2.9%, P = 0.0063). After 24 Gy irradiation, Fancd2(-/-) mice had a higher oral cavity percentage of tongue ulceration compared to Fancd2(+/+) mice irradiated with higher doses of 26 Gy (P = 0.0123). Baseline and postirradiation oral cavity gene transcripts were altered in Fancd2(-/-) mice compared to Fancd2(+/+) controls. Fancd2(-/-) mice had decreased baseline femur marrow CFU-GM, BFUe and CFU-GEMM, which further decreased after 24 or 26 Gy head and neck irradiation. These changes were not seen in head- and neck-irradiated Fancd2(+/+) mice. In radiosensitive Fancd2(-/-) mice, biomarkers of both local oral cavity and distant marrow radiation toxicity were ameliorated by intraoral JP4-039/F15. We propose that Fancd2(-/-) mice are a valuable radiosensitive animal model system, which can be used to evaluate potential radioprotective agents.

[Effects of ectomesenchymal stem cells on hematopoiesis after total body irradiation].

OBJECTIVE: To investigate the effects of ectomesenchymal stem cells on hematopoiesis after total body irradiation in rats. METHODS: The primary ectomesenchymal stem cells were isolated from E11.5 SD fetal mandibular processes by 2.5 g/L trypsin and cultured with DMEM/F12. The morphology and growth rate were observed by inverted microscope. Eighty SD male rats randomly divided into ectomesenchymal stem cells group (n = 20), fibroblast group (n = 20), saline group (n = 20) and control group (n = 20), the first three groups were irradiated with 60Co gamma rays at 6. 0 Gy. The number of their bone marrow nucleated cells was counted after 4 weeks; the forming ability of colony-forming unit-granulocyte macrophage (CFU-GM) and histopathology of bone marrow were also observed. RESULTS: The cultured cells displayed monolayer growth and fibroblast-like with 2-4 processes. The ectomesenchymal stem cells could increase the number of bone marrow nucleated cells and peripheral blood white cell count, and improve the forming ability of CFU-GM. After 4 weeks of transplantation, the number of the peripheral blood white cells in group A was more than that in groups B and C (P < 0.05), the contents of Hb in groups A and D was significantly higher than those in groups B and C (P < 0.05). After 4 weeks, the bone marrow nucleated cells in group A were significant more than those in groups B and C (P < 0.01); CFU-GM in groups A and D was higher than that in groups B and C (P < 0.01). CONCLUSION: Ectomesenchymal stem cells have characteristics of stem cells. It may improve hematopoiesis recovery of irradiated rats.

In vivo effects of Mpl ligand administration and emerging clinical applications for the Mpl ligands.

The Mpl ligands are a family of closely related hematopoietic growth factors that bind to the thrombopoietin receptor, c-Mpl. In addition to the endogenous Mpl ligand, thrombopoietin, two recombinant Mpl ligands, recombinant thrombopoietin and pegylated megakaryocyte growth and development factor (PEG-MGDF) are under investigation. Endogenous thrombopoietin regulates most of the normal production of platelets but also is essential for the normal development of other lineages. When recombinant thrombopoietin or PEG-MGDF is administered to normal animals or humans, there is a dose-dependent increase in the platelet count but no effect on leukocytes or erythrocytes. When administered following chemotherapy in animal models or humans, Mpl ligands reduce the duration and sometimes the degree of thrombocytopenia. The Mpl ligands also may be effective in reducing the thrombocytopenia of patients with HIV infection, liver disease, myelodysplasia, or after plateletpheresis.

Stable mixed hematopoietic chimerism in DLA-identical littermate dogs given sublethal total body irradiation before and pharmacological immunosuppression after marrow transplantation.

Postgrafting cyclosporine (CSP) given for 35 days resulted in establishment of stable marrow grafts from DLA-identical canine littermates after otherwise suboptimal, but nevertheless, lethal conditioning with 450 cGy of total body irradiation (TBI). We now asked whether sustained allografts could be achieved after sublethal TBI or without TBI. Five groups of recipients were studied. Dogs in group 1 were given 200 cGy TBI and postgrafting CSP, 15 mg/kg twice daily by mouth on days -1 to 35 posttransplant. Dogs in group 2 were given 200 cGy TBI and methotrexate (MTX), 0.4 mg/kg intravenously (I.V.) on days 1, 3, 6, and 11 along with CSP. Dogs in group 3 were given 200 cGy TBI and CSP along with mycophenolate mofetil (MMF), 10 mg/kg twice daily subcutaneously (S.C.) on days 0 to 27 after transplant, a novel immunosuppressive combination. Dogs in group 4 were given 100 cGy TBI and MMF/CSP. Dogs in group 5 were not given TBI and they received MMF/CSP posttransplant. Allografts were assessed by (Ca)n dinucleotide repeat polymorphism studies in cells from peripheral blood, lymph nodes, and marrow. Dogs in group 1 had transient mixed donor-host hematopoietic chimerism for no more than 4 weeks. Three of six dogs in group 2 had transient mixed chimerism for 3 to 11 weeks, and three have remained stable mixed chimeras for up to 60 weeks now. Four of five dogs in group 3 have remained stable mixed chimeras for 54 to 57 weeks now, while one lost the allograft after 12 weeks. All dogs in groups 4 and 5 rejected their allografts after 2 to 12 weeks. In summary, the establishment of stable mixed hematopoietic chimerism following nonmyelosuppressive and nontoxic conditioning programs has remained a difficult goal. Here we present evidence in a large random-bred animal species that this goal may be achievable with pharmacological immunosuppression postgrafting, capable of inhibiting both host-versus-graft (HVG) and graft-versus-host (GVH) reactions in the setting of DLA-identical grafts.

A single dose of pegylated leridistim significantly improves neutrophil recovery in sublethally irradiated rhesus macaques.

Leridistim, a member of the myelopoietin family of dual receptor agonists that binds interleukin-3 and G-CSF receptors, has been shown to enhance hematopoietic activity in rhesus monkeys above that observed with either cytokine alone or in combination. This study demonstrated the ability of a pegylated form of leridistim (peg-leridistim), administered s.c., as a single- or two-dose regimen separated by 4 or 7 days, to significantly improve neutrophil recovery following radiation-induced myelosuppression. Rhesus macaques were total body x-irradiated (250 kVp, TBI) to 600 cGy. Following TBI, two groups received peg-leridistim (n = 5) or leridistim (n = 4) at a dose of 600 microg/kg on day 1, while two other groups (both n = 4) received peg-leridistim at 200 microg/kg on day 1 and day 4, or day 1 and day 7. The irradiation controls (n = 7) received 0.1% autologous serum for 18 days. All peg-leridistim treatment schedules significantly improved all neutrophil-related parameters following TBI as compared with nontreated controls and were equivalent in effect when compared among themselves. Administration of a single high dose or two separate lower doses of peg-leridistim significantly improved neutrophil regeneration, in a manner equal to that of conventional daily or abbreviated every-other-day administration of leridistim in this nonhuman primate model of severe myelosuppression.