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In recent years, multipotent mesenchymal stromal cells (MSCs) have demonstrated tremendous potential for use in regenerative medicine. CXCR4, the receptor for CXCL12, is highly expressed by bone marrow (BM) MSCs and the CXCR4/CXCL12 axis has been shown to be important for migration and homing of BM-MSCs. Typically, MSCs used for clinical applications are collected after culture expansion using enzymatic methods, such as trypsin. Here, we compared different commercially available enzymatic and non-enzymatic methods for collection and dissociation of MSCs from culture plastics and their effects on CXCR4 expression by MSCs. We found that whereas non-enzymatic dissociation buffers and methods maintained CXCR4 expression, all tested enzymatic dissociation solutions dramatically decreased expression of CXCR4. We, therefore, strongly recommend the use of non-enzymatic dissociation methods, followed by filtration through a cell strainer to obtain single cell suspensions, in order to preserve maximal CXCR4 expression and optimal homing of cells.

The newly developed 6-hydroxychromanol derivate SUL-109 was shown to provide protection during hypothermic storage of several cell lines, but has not been evaluated in hematopoietic stem cells (HSCs). Hypothermic preservation of HSCs would be preferred over short-term cryopreservation to prevent cell loss during freezing/thawing and would be particularly useful for short-term storage, such as during conditioning of patients or transport of HSC transplants. Here we cultured human CD34+ umbilical cord blood (UCB) cells and lineage-depleted (Lin–) Balb/c bone marrow (BM) cells for up to 7 days in serum-free HSC expansion medium with hematopoietic growth factors. SUL-109-containing cultures were stored at 4°C for 3 to 14 days. The UCB cells were tested for viability, cell cycle, and reactive oxygen species (ROS). DMSO-cryopreserved Lin– BM cells or Lin– BM cells maintained for 14 days at 4°C were transplanted into RAG2−/− Balb/c mice and engraftment was followed for 6 months. The addition of SUL-109 during the hypothermic storage of expanded CD34+ UCB cells provided a significant improvement in cell survival of the immature CD34+/CD38- fraction after 7 days of hypothermic storage through scavenging of hypothermia-induced ROS and was able to preserve the multilineage capacity of human CD34+ UCB cells for up to 14 days of cold storage. In addition, SUL-109 protected murine BM Lin– cells from 14 days of hypothermic preservation and maintained their engraftment potential after transplantation in immune-deficient RAG2−/− mice. Our data indicate that SUL-109 is a promising novel chemical for use as a protective agent during cold storage of human and murine HSCs to prevent hypothermia-induced apoptosis and promote cell viability.

Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition., Graphical Abstract, This publication reports that stem cell gene therapy using a codon-optimized gene encoding acid α-glucosidase (GAA) cures the mouse model of Pompe disease, a lysosomal storage disorder.

Purpose 5-Androstene-3β,17β-diol (5-AED) stimulates recovery of hematopoiesis after exposure to radiation. To elucidate its cellular targets, the effects of 5-AED alone and in combination with (pegylated) granulocyte colony-stimulating factor and thrombopoietin (TPO) on immature hematopoietic progenitor cells were evaluated following total body irradiation. Methods and Materials BALB/c mice were exposed to radiation delivered as a single or as a fractionated dose, and recovery of bone marrow progenitors and peripheral blood parameters was assessed. Results BALB/c mice treated with 5-AED displayed accelerated multilineage blood cell recovery and elevated bone marrow (BM) cellularity and numbers of progenitor cells. The spleen colony-forming unit (CFU-S) assay, representing the life-saving short-term repopulating cells in BM of irradiated donor mice revealed that combined treatment with 5-AED plus TPO resulted in a 20.1-fold increase in CFU-S relative to that of placebo controls, and a 3.7 and 3.1-fold increase in comparison to 5-AED and TPO, whereas no effect was seen of Peg-G-CSF with or without 5-AED. Contrary to TPO, 5-AED also stimulated reconstitution of the more immature marrow repopulating (MRA) cells. Conclusions 5-AED potently counteracts the hematopoietic effects of radiation-induced myelosuppression and promotes multilineage reconstitution by stimulating immature bone marrow cells in a pattern distinct from, but synergistic with TPO.

Clinical trials have demonstrated the potential of ex vivo hematopoietic stem cell gene therapy to treat X-linked severe combined immunodeficiency (SCID-X1) using retroviral vectors, leading to immune system functionality in the majority of treated patients without pretransplant conditioning. The success was tempered by insertional oncogenesis in a proportion of the patients. To reduce the genotoxicity risk, a self-inactivating (SIN) lentiviral vector (LV) with improved expression of a codon optimized human interleukin-2 receptor gamma gene (IL2RG) cDNA (co gamma c), regulated by its 1.1 kb promoter region (gamma cPr), was compared in efficacy to the viral spleen focus forming virus (SF) and the cellular phosphoglycerate kinase (PGK) promoters. Pretransplant conditioning of IL2rg(-/-) mice resulted in long-term reconstitution of T and B lymphocytes, normalized natural antibody titers, humoral immune responses, ConA/IL-2 stimulated spleen cell proliferation, and polyclonal T-cell receptor gene rearrangements with a clear integration preference of the SF vector for proto-oncogenes, contrary to the PGK and gamma cPr vectors. We conclude that SIN lentiviral gene therapy using co gamma c driven by the gamma cPr or PGK promoter corrects the SCID phenotype, potentially with an improved safety profile, and that low-dose conditioning proved essential for immune competence, allowing for a reduced threshold of cell numbers required. Received 23 February 2011; accepted 31 May 2011; published online 12 July 2011. doi:10.1038/mt.2011.127

Pompe disease (acid α-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector–mediated expression of human acid α-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid α-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid α-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.

It is a longstanding question which bone marrow–derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34+CD1a– and CD34+CD1a+ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34+CD1a– thymocytes but not in CD34+CD1a+ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34+CD1a– subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34+CD1a– thymocytes toward the T-cell lineage, as shown by T-cell receptor δ gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34+CD1a+ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34+CD1a– and CD34+CD1a+ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity.

Summary. Thrombopoietin (TPO) immunogenicity hampers its development as a therapeutic agent for attenuating thrombocytopenia and improving platelet harvest in donors. This work was aimed at validating, in mouse and in monkey experiments, a thrombopoiesis computer-model prediction that platelet counts, similar to those obtained with accepted TPO dose scheduling, can also be achieved by new and safer schedules of significantly reduced doses. To this end we compared, in a two-arm mouse experiment, platelet increases obtained with a single intraperitoneal dosing of recombinant mouse TPO (17·5 μg/kg), with those obtained by the model-suggested protocol of a significantly reduced dose (2 μg/kg on 4 consecutive days). The two TPO regimens generated similar platelet profiles, peaking at ca. 2700 × 109/l platelets. In rhesus monkeys, treated by rhesus monkey recombinant TPO (5 μg/kg on 4 consecutive days), the suggested protocol yielded effective platelet stimulation with significantly reduced immunogenicity. The model's ability to predict individual monkey responses to several new TPO administration protocols was further validated, proving sufficient robustness in providing good predictions with limited input data. The simulation tool could be used for testing the effects of different therapeutic agents on thrombopoiesis. Human trials are warranted for testing the suggested improved TPO protocol, possibly in conjunction with chemotherapy.

Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by similar to 6-fold and similar to 10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by similar to 17-fold and similar to 32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1(+), c-Kit(+) HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy.

Thrombopoietin (TPO) has been used in preclinical myelosuppression models to evaluate the effect on hematopoietic reconstitution. Here we report the importance of dose and dose scheduling for multilineage reconstitution after myelosuppressive total body irradiation (TBI) in mice. After 6 Gy TBI, a dose of 0.3 μg TPO/mouse (12 μg/kg) intraperitoneally (IP), 0 to 4 hours after TBI, prevented the severe thrombopenia observed in control mice, and in addition stimulated red and white blood cell regeneration. Time course studies showed a gradual decline in efficacy after an optimum within the first hours after TBI, accompanied by a replacement of the multilineage effects by lineage dominant thrombopoietic stimulation. Pharmacokinetic data showed that IP injection resulted in maximum plasma levels 2 hours after administration. On the basis of the data, we inferred that a substantial level of TPO was required at a critical time interval after TBI to induce multilineage stimulation of residual bone marrow cells. A more precise estimate of the effect of dose and dose timing was provided by intravenous administration of TPO, which showed an optimum immediately after TBI and a sharp decline in efficacy between a dose of 0.1 μg/mouse (4 μg/kg; plasma level 60 ng/mL), which was fully effective, and a dose of 0.03 μg/mouse (1.2 μg/kg; plasma level 20 ng/mL), which was largely ineffective. This is consistent with a threshold level of TPO required to overcome initial c-mpl–mediated clearance and to reach sufficient plasma levels for a maximum hematopoietic response. In mice exposed to fractionated TBI (3 × 3 Gy, 24 hours apart), IP administration of 0.3 μg TPO 2 hours after each fraction completely prevented the severe thrombopenia and anemia that occurred in control mice. Using short-term transplantation assays, ie, colony-forming unit–spleen (CFU-S) day 13 (CFU-S-13) and the more immature cells with marrow repopulating ability (MRA), it could be shown that TPO promoted CFU-S-13 and transiently depleted MRA. The initial depletion of MRA in response to TPO was replenished during long-term reconstitution followed for a period of 3 months. Apart from demonstrating again that MRA cells and CFU-S-13 are separate functional entities, the data thus showed that TPO promotes short-term multilineage repopulating cells at the expense of more immature ancestral cells, thereby preventing pancytopenia. The short time interval available after TBI to exert these effects shows that TPO is able to intervene in mechanisms that result in functional depletion of its multilineage target cells shortly after TBI and emphasizes the requirement of dose scheduling of TPO in keeping with these mechanisms to obtain optimal clinical efficacy. © 1998 by The American Society of Hematology.

The further improvement of gene transfer into hematopoietic stem cells and their direct progeny will be greatly facilitated by markers that allow rapid detection and efficient selection of successfully transduced cells. For this purpose, a retroviral vector was designed and tested encoding a recombinant version of the Aequorea victoria green fluorescent protein that is enhanced for high-level expression in mammalian cells (EGFP). Murine cell lines (NIH 3T3, Rat2) and bone marrow cells transduced with this retroviral vector demonstrated a stable green fluorescence signal readily detectable by flow cytometry. Functional analysis of the retrovirally transduced bone marrow cells showed EGFP expression in in vitro clonogenic progenitors (GM-CFU), day 13 colony-forming unit-spleen (CFU-S), and in peripheral blood cells and marrow repopulating cells of transplanted mice. In conjunction with fluorescence-activated cell sorting (FACS) techniques EGFP expression could be used as a marker to select for greater than 95% pure populations of transduced cells and to phenotypically define the transduced cells using antibodies directed against specific cell-surface antigens. Detrimental effects of EGFP expression were not observed: fluorescence intensity appeared to be stable and hematopoietic cell growth was not impaired. The data show the feasibility of using EGFP as a convenient and rapid reporter to monitor retroviral-mediated gene transfer and expression in hematopoietic cells, to select for the genetically modified cells, and to track these cells and their progeny both in vitro and in vivo.

Recombination activating gene 2 (RAG2) deficiency results in severe combined immunodeficiency (SCID) with complete lack of T and B lymphocytes. Initial gammaretroviral gene therapy trials for other types of SCID proved effective, but also revealed the necessity of safe vector design. We report the development of lentiviral vectors with the spleen focus forming virus (SF) promoter driving codon-optimized human RAG2 (RAG2co), which improved phenotype amelioration compared to native RAG2 in Rag2(-/-) mice. With the RAG2co therapeutic transgene, T-cell receptor (TCR) and immunoglobulin repertoire, T-cell mitogen responses, plasma immunoglobulin levels and T-cell dependent and independent specific antibody responses were restored. However, the thymus double positive T-cell population remained subnormal, possibly due to the SF virus derived element being sensitive to methylation/silencing in the thymus, which was prevented by replacing the SF promoter by the previously reported silencing resistant element (ubiquitous chromatin opening element (UCOE)), and also improved B-cell reconstitution to eventually near normal levels. Weak cellular promoters were effective in T-cell reconstitution, but deficient in B-cell reconstitution. We conclude that immune functions are corrected in Rag2(-/-) mice by genetic modification of stem cells using the UCOE driven codon-optimized RAG2, providing a valid optional vector for clinical implementation.

Thrombopoietin (TPO) immunogenicity hampers its development as a therapeutic agent for attenuating thrombocytopenia and improving platelet harvest in donors. This work was aimed at validating, in mouse and in monkey experiments, a thrombopoiesis computer-model prediction that platelet counts, similar to those obtained with accepted TPO dose scheduling, can also be achieved by new and safer schedules of significantly reduced doses. To this end we compared, in a two-arm mouse experiment, platelet increases obtained with a single intraperitoneal dosing of recombinant mouse TPO (17.5 microg/kg), with those obtained by the model-suggested protocol of a significantly reduced dose (2 microg/kg on 4 consecutive days). The two TPO regimens generated similar platelet profiles, peaking at ca. 2700 x 10(9)/l platelets. In rhesus monkeys, treated by rhesus monkey recombinant TPO (5 microg/kg on 4 consecutive days), the suggested protocol yielded effective platelet stimulation with significantly reduced immunogenicity. The model's ability to predict individual monkey responses to several new TPO administration protocols was further validated, proving sufficient robustness in providing good predictions with limited input data. The simulation tool could be used for testing the effects of different therapeutic agents on thrombopoiesis. Human trials are warranted for testing the suggested improved TPO protocol, possibly in conjunction with chemotherapy.

Purpose: The therapeutic potential of thrombopoietin (TPO), the major regulator of platelet production, was evaluated for hematopoietic recovery and survival in mice following lethal and supralethal total body irradiation (TBI). Methods and Materials: Hematopoietic recovery was studied in C57BL6/J mice after 8 Gy TBI (gamma-rays). Survival experiments were performed with C57BL6/J and BCBA F 1 mice. Two protocols of TPO administration were evaluated: treatment for 7 consecutive days (7 × 0.3 μg/mice) beginning 2 h after exposure, or a single dose (0.3 μg/mice) administered 2 h after irradiation. Results: TPO improved the platelet nadir and accelerated the platelet reconstitution of irradiated mice in comparison to placebo-treated mice. Recovery of neutrophils and erythrocytes was stimulated as well. TPO induced an accelerated recovery of hematopoietic progenitors and immature multilineage progenitors in bone marrow and spleen. In addition, TPO administration induced approximately 90% survival of 8 Gy irradiated C57BL6/J mice, a TBI dose which resulted in 100% mortality within 30 days for placebo-treated mice. Single TPO administration was as effective as repeated injections for hematopoietic recovery and prevention of mortality. Dose-effect survival experiments were performed in BCBA F 1 mice and demonstrated that TPO shifted the LD 50/30 from approximately 9.5 Gy to 10.5 Gy TBI given as a single dose, and from 14 Gy to as high as 17 Gy when TBI was given in three equal doses, each separated by 24 h. Conclusion: These results demonstrate that the multilineage hematopoietic effects of TPO may be advantageously used to protect against lethal bone marrow failure following high dose TBI.

Thrombopoietin (TPO) has been used in preclinical myelosuppression models to evaluate the effect on hematopoietic reconstitution. Here we report the importance of dose and dose scheduling for multilineage reconstitution after myelosuppressive total body irradiation (TBI) in mice. After 6 Gy TBI, a dose of 0.3 μg TPO/mouse (12 μg/kg) intraperitoneally (IP), 0 to 4 hours after TBI, prevented the severe thrombopenia observed in control mice, and in addition stimulated red and white blood cell regeneration. Time course studies showed a gradual decline in efficacy after an optimum within the first hours after TBI, accompanied by a replacement of the multilineage effects by lineage dominant thrombopoietic stimulation. Pharmacokinetic data showed that IP injection resulted in maximum plasma levels 2 hours after administration. On the basis of the data, we inferred that a substantial level of TPO was required at a critical time interval after TBI to induce multilineage stimulation of residual bone marrow cells. A more precise estimate of the effect of dose and dose timing was provided by intravenous administration of TPO, which showed an optimum immediately after TBI and a sharp decline in efficacy between a dose of 0.1 μg/mouse (4 μg/kg; plasma level 60 ng/mL), which was fully effective, and a dose of 0.03 μg/mouse (1.2 μg/kg; plasma level 20 ng/mL), which was largely ineffective. This is consistent with a threshold level of TPO required to overcome initial c-mpl–mediated clearance and to reach sufficient plasma levels for a maximum hematopoietic response. In mice exposed to fractionated TBI (3 × 3 Gy, 24 hours apart), IP administration of 0.3 μg TPO 2 hours after each fraction completely prevented the severe thrombopenia and anemia that occurred in control mice. Using short-term transplantation assays, ie, colony-forming unit–spleen (CFU-S) day 13 (CFU-S-13) and the more immature cells with marrow repopulating ability (MRA), it could be shown that TPO promoted CFU-S-13 and transiently depleted MRA. The initial depletion of MRA in response to TPO was replenished during long-term reconstitution followed for a period of 3 months. Apart from demonstrating again that MRA cells and CFU-S-13 are separate functional entities, the data thus showed that TPO promotes short-term multilineage repopulating cells at the expense of more immature ancestral cells, thereby preventing pancytopenia. The short time interval available after TBI to exert these effects shows that TPO is able to intervene in mechanisms that result in functional depletion of its multilineage target cells shortly after TBI and emphasizes the requirement of dose scheduling of TPO in keeping with these mechanisms to obtain optimal clinical efficacy. © 1998 by The American Society of Hematology.

Retroviral vectors are widely used for the introduction of exogenous genetic material into hematopoietic cells. Here we report the generation of retroviral vectors containing the Aequorea victoria green fluorescent protein (GFP) gene and improved versions thereof. Murine fibroblasts transduced with the mutant GFP genes demonstrated a distinct green fluorescent signal in fluorescence-activated cell sorter (FACS) analysis. The relative intensities of peak green fluorescence observed with different GFP mutants were in the order EGFP>hGFP(S65T)> GFP-PTS1 or RSGFP>wildtype GFP (wtGFP). Furthermore, GFP-PTS1 expression was observed in murine (3T3, Rat2, and freshly-cultured bone marrow) and human (K562) cells transduced with the corresponding retroviral vector. The GFP-PTS1 positive phenotype could be selected for by FACS and appeared to be stable for at least 1 month in murine fibroblasts and human K562 cells. Therefore, these GFP variants are convenient selectable markers to monitor retroviral-mediated gene transfer and expression in mammalian hematopoietic cells.

Flt3/flk2 belongs to the class III hematopoietic growth factor receptors with intrinsic kinase activity, ligand-activated tyrosine phosphorylation and a central role in early stages of hematopoiesis. Flt3-ligand (FL) has been shown to enhance the effects of several growth factors, including Granulocyte-colony stimulating factor (G-CSF). In this study the reconstitution ability of FL+G-CSF primed CD34+ cells was evaluated in a nonhuman primate model of radiation-induced myeloablative treatment followed by transplantation of stimulated autologous bone marrow (BM) and/or peripheral blood (PB) cells. Rhesus monkeys (n=9) were subjected to subcutaneous injections with FL (100 μg/kg/day) and G-CSF (100 μg/kg/day) for 7 to 14 days and subsequently irradiated with a lethal dose of total body irradiation (TBI) followed by transplantation with primed stem cells or with a midlethal dose of TBI (n=2) without transplantation. White blood cell counts (WBC) in PB showed a steep increase and reached a mean level of 98.9×109/L (71.5–131.7×109/L) after 7 days (P

5-AED (5-androstene-3β,17β-diol) is a naturally occurring adrenal cortical steroid, which displays radioprotective effects in both rodents and non-human primates, resulting in accelerated multilineage hematopoiesis and enhanced survival after total body irradiation (TBI), including a 1-log accelerated CD34+ cell reconstitution in bone marrow of non-human primates. Pegylated granulocyte-colony stimulating factor (Peg-G-CSF) is known to stimulate lineage-specific recovery of neutrophils, whereas the effects of thrombopoietin (TPO) are broader and include protection of short-term spleen repopulating immature cells as well as platelet recovery. To gain insight into the mechanism of 5-AED on immature hematopoietic cells, the effects of 5-AED on multilineage hematopoiesis and recovery of specific repopulating stem and progenitor cell subsets after TBI was evaluated in combination with and relative to either Peg-G-CSF or TPO. For direct measurements of the radioprotective effect of 5-AED, BALB/c mice were exposed to a midlethal dose of 6 Gy TBI. Two hours after TBI, mice were injected IM with 40 mg/kg 5-AED or the carrier, with or without 0.225 mg TPO or 10 mg Peg-G-CSF IP. Radioprotective effects of 5-AED on immature repopulating cell subsets were assessed by exposing BALB/c donor mice to 3 fractions of 2 Gy TBI, separated by 24 hours, and treatment with 40 mg/kg/d 5-AED or the carrier IM, or 0.7 mg TPO IP after each fraction or a single injection of 10 mg Peg-G-CSF IP after the first fraction. Twenty four hours after the last fraction, bone marrow of donor mice was examined for immature cell content per femur using the marrow repopulating ability (MRA day 13) assay and the CFU-S day 12 after transplantation in 8 Gy irradiated mice. After 6 Gy TBI, BALB/c mice treated with 5-AED displayed an accelerated multilineage recovery with increased white blood cells (p

5-androstene-3β,17β-diol, a naturally occurring adrenal steroid, has been shown to protect mice exposed to γ ray total body irradiation (TBI), as apparent by enhanced survival, stimulated myelopoiesis and elevated nadirs of neutrophils and platelets. In the present study, rhesus monkeys (Macaca mulatta; n=8) were subjected to the midlethal dose of 6 Gy 6 MV TBI at a dose rate of 35 cGy/minute, after which 4 randomly selected monkeys (2 males and 2 females) received 15 mg/kg IM of the testing compound HE2100 for 5 consecutive days, starting at 2 hours after irradiation. Placebo monkeys (also 2 males, 2 females) received the carrier IM for 5 days. TBI resulted in profound pancytopenia in all monkeys, which lasted 3 to 4 weeks in the placebo control monkeys. However, treatment with HE2100 reduced the period of severe neutropenia, with an 8 days earlier neutrophil recovery to levels >0.5x109/L than the placebo group, i.e. 12.8 days (range 11–15) vs 20.8 days (range 17–22), respectively. The neutrophil recovery was also reflected by accelerated recovery of CD11b+ cells. CD16/56+NK cells trailed the neutrophils. Recovery of reticulocytes was markedly enhanced in the HE2100 group to reach levels of >1% in peripheral blood (PB) by day 18.8 (range 18–20), whereas placebo control monkeys did not reach this level until day 26.3 (range 23–32). Reticulocyte reconstitution was accompanied by a transient erythroblastosis in HE2100 monkeys, emphasizing the pronounced stimulation of red cell reconstitution. A most prominent effect of HE2100 was noted for platelet recovery, since HE2100 effectively counteracted the need for transfusions, with only 1 (range 0–2) transfusion needed to maintain platelets at the used high threshold level of >40x109/L as apposed to 4 (range 2–8) in the placebo controls. The reconstitution of PB cells was preceded by a markedly accelerated recovery of immature bone marrow (BM) cells in HE2100 treated monkeys. At 2 weeks post-TBI, numbers of BM CD34+ cells approached pre-irradiation levels, whereas placebo monkeys did not reach such levels before the 4th week after TBI. Consequently, CD34+ cells in BM of HE2100 monkeys differed as much as 2 logs from those in placebo controls at 2 weeks after TBI, which was also reflected in the clonogenic progenitor cells GM-CFU and BFU-E, in accelerated normalization of BM cellularity and in PB CD34+ levels. Direct local or systemic toxic effects were not observed during administration of the steroid, but HE2100 monkeys displayed an increase of, on average, 10% in body weight due to fluid retention starting in the 2nd week after TBI, resulting in transient edema, which resolved without sequelae. This preclinical study characterizes HE2100 as a most potent novel agent to promote stem cell reconstitution and multilineage myelopoiesis after cytoreductive radiation exposure, resulting in enhanced reticulocyte, neutrophil and platelet recovery and profoundly decreased needs for transfusions and antibiotic treatment. The mechanisms involved are subject of further analyses.

Replication defective retroviruses deliver transgenes into hematopoietic stem cells (HSC) with high efficacy. The first successful clinical study revealed an unexpectedly high frequency of insertional leukemogenesis, not predicted by extensive preclinical experimental studies. In cell lines, the frequency of insertional oncogenic events has been estimated at approximately 10-7 per transduced cell. To examine the in vivo incidence, we monitored mice transplanted with transduced stem cells for observation times up to 342 days after transplantation and retransplanted the cells into secondary recipients at selected time intervals. The observations were made in the context of ectopic expression of wtSTAT5 to study its effect on stem cell repopulating capacity.

Graft-versus-host disease (GvHD) after bone-marrow transplantation in dogs is controlled by many different genetic systems. In littermate combinations identical for the major histocompatibility complex (MHC) the number of systems that influence GvHD is related to the number of donor lymphocytes injected. If the number of donor lymphocytes administered is sufficiently low, minor histocompatibility systems do not influence survival after bone-marrow transplantation. With increasing numbers of donor lymphocytes the beneficial influence of MHC matching on GvH incidence and severity disappears and minor histocompatibility antigens, coded for on at least two other autosomal chromosomes as well as possibly the Y chromosome, can cause severe GvHD. In contrast, the X chromosome does not appear to carry a histocompatibility system that is of relevance to GvHD control. The severity and tissue distribution of histological signs of GvHD in recipients of bone-marrow and lymph-node cells from MHC-identical donors are similar to those in recipients of MHC-mismatched bone-marrow cells. Female donors do appear to cause severe GvHD more frequently than males. In contrast to rhesus monkey and human bone-marrow cells, dog bone-marrow cells are negative in PHA tests. This is in accordance with the generally benign course of GvHD in dogs that are treated with bone-marrow cells only from histocompatible littermate donors. The influence of the sex of the bone-marrow donor on GvHD incidence and severity is not reflected in differences between PHA tests with male and female dog lymphocytes. A better predictive test for GvH potential than the PHA test appears to be needed. Alternatives to additional donor selection for the prevention of GvHD in histocompatible recipients appear to be the use of a male donor and the removal of lymphocytes from bone-marrow-cell suspensions prior to transplantation.