Fetal Stem Cell Transplantation Research Articles

Maternal administration of busulfan before in utero transplantation of human hematopoietic stem cells enhances engraftments in sheep

In utero transplantation (IUT) of human hematopoietic stem cells has been conducted in sheep, which are used as large animal models of human hematopoietic reconstitution and models for clinical IUT; however, the levels of engraftment have generally been low. Busulfan (BU), a myeloablative agent, is often administered to patients before hematopoietic stem cells transplantation to improve the engraftment. In this study, hematopoietic activity was evaluated in adult sheep after administering BU at different doses. Next, pregnant ewes were administered BU, and dams as well as their fetuses were evaluated, as BU readily crosses the sheep placenta. Then, the BU dose with the desired outcomes was selected and administered to pregnant ewes at 2 or 6 days before performing IUT using human cord blood CD34(+) cells. The engraftment was evaluated in recipients that underwent IUT in the presence or absence of BU. As a result, hematopoietic activity was safely and transiently suppressed in adult sheep treated with 5 to 7.5 mg/kg BU. BU crossed the sheep placenta, and fetal sheep were indeed conditioned by administering 3 mg/kg BU to pregnant ewes. Engraftment of human CD34(+) cells in fetal recipients was enhanced when IUT was carried out 6 days post-BU. Up to 3.3% engraftment levels (in terms of bone marrow colony-forming units) were achieved with the IUT of 0.72 to 2.4 million CD34(+) cells when BU was used. BU can be administered to pregnant ewes to effectively condition the fetal recipient for IUT with enhanced engraftment of donor cells.

Upregulating CXCR4 in human fetal mesenchymal stem cells enhances engraftment and bone mechanics in a mouse model of osteogenesis imperfecta.

Stem cells have considerable potential to repair damaged organs and tissues. We previously showed that prenatal transplantation of human first trimester fetal blood mesenchymal stem cells (hfMSCs) in a mouse model of osteogenesis imperfecta (oim mice) led to a phenotypic improvement, with a marked decrease in fracture rate. Donor cells differentiated into mature osteoblasts, producing bone proteins and minerals, including collagen type Iα2, which is absent in nontransplanted mice. This led to modifications of the bone matrix and subsequent decrease of bone brittleness, indicating that grafted cells directly contribute to improvement of bone mechanical properties. Nevertheless, the therapeutic effect was incomplete, attributing to the limited level of engraftment in bone. In this study, we show that although migration of hfMSCs to bone and bone marrow is CXCR4-SDF1 (SDF1 is stromal-derived factor) dependent, only a small number of cells present CXCR4 on the cell surface despite high levels of internal CXCR4. Priming with SDF1, however, upregulates CXCR4 to increase the CXCR4(+) cell fraction, improving chemotaxis in vitro and enhancing engraftment in vivo at least threefold in both oim and wild-type bone and bone marrow. Higher engraftment in oim bones was associated with decreased bone brittleness. This strategy represents a step to improve the therapeutic benefits of fetal cell therapy toward being curative.

Stem Cell Therapy Extends Incubation and Survival Time in Prion-Infected Mice in a Time Window–Dependant Manner

Prion diseases, which are mostly represented in humans by Creutzfeldt-Jakob disease, are transmissible neurodegenerative disorders characterized by vacuolization and neuronal loss, as well as by the accumulation of an abnormal form of the prion protein. These disorders have yet no effective treatment, and drugs that block prion replication in vitro do not significantly slow down the progression of the disease when used in vivo at late stages. Cell therapy that has been already tested in other neurodegenerative disorders therefore represents an interesting alternative approach. In this study, we showed for the first time in prion diseases that intracerebral transplantation of fetal neural stem cells significantly extended both incubation and survival time. This result was dependant on the time window chosen for the engraftment and was obtained with both genetically modified and wild-type stem cells, therefore forging a path toward efficient stem cell therapy for human prion diseases.

Is Human Fetal Liver Stem Cell Transplantation a Panacea for Alcohol-Induced Liver Decompensation?

Is Human Fetal Liver Stem Cell Transplantation a Panacea for Alcohol-Induced Liver Decompensation?

Graft‐induced dyskinesias in Parkinson's disease: High striatal serotonin/dopamine transporter ratio

AbstractGraft‐induced dyskinesias are a serious complication after neural transplantation in Parkinson's disease. One patient with Parkinson's disease, treated with fetal grafts 14 years ago and deep brain stimulation 6 years ago, showed marked improvement of motor symptoms but continued to suffer from OFF‐medication graft‐induced dyskinesias. The patient received a series of clinical and imaging assessments. Positron emission tomography and single‐photon emission computed tomography 14 years posttransplantation revealed an elevated serotonin/dopamine transporter ratio in the grafted striatum compatible with serotonergic hyperinnervation. Inhibition of serotonin neuron activity by systemic administration of a 5‐HT1A agonist suppressed graft‐induced dyskinesias. Our data provide further evidence that serotonergic neurons mediate graft‐induced dyskinesias in Parkinson's disease. Achieving a normal striatal serotonin/dopamine transporter ratio following transplantation of fetal tissue or stem cells should be necessary to avoid the development of graft‐induced dyskinesias. © 2011 Movement Disorder Society

Transplantation of human fetal blood stem cells in the osteogenesis imperfecta mouse leads to improvement in multiscale tissue properties

AbstractOsteogenesis imperfecta (OI or brittle bone disease) is a disorder of connective tissues caused by mutations in the collagen genes. We previously showed that intrauterine transplantation of human blood fetal stem/stromal cells in OI mice (oim) resulted in a significant reduction of bone fracture. This work examines the cellular mechanisms and mechanical bone modifications underlying these therapeutic effects, particularly examining the direct effects of donor collagen expression on bone material properties. In this study, we found an 84% reduction in femoral fractures in transplanted oim mice. Fetal blood stem/stromal cells engrafted in bones, differentiated into mature osteoblasts, expressed osteocalcin, and produced COL1a2 protein, which is absent in oim mice. The presence of normal collagen decreased hydroxyproline content in bones, altered the apatite crystal structure, increased the bone matrix stiffness, and reduced bone brittleness. In conclusion, expression of normal collagen from mature osteoblast of donor origin significantly decreased bone brittleness by improving the mechanical integrity of the bone at the molecular, tissue, and whole bone levels.

Human Fetal Liver-Derived Stem Cell Transplantation as Supportive Modality in the Management of End-Stage Decompensated Liver Cirrhosis

Liver transplantation is the only existing modality for treating decompensated liver cirrhosis. Several factors, such as nonavailability of donors, combined with operative risks, complications associated with rejection, usage of immunosuppressive agents, and cost intensiveness, make this strategy available to only a few people. With a tremendous upsurge in the mortality rate of patients with liver disorders worldwide, there is a need to search for an alternative therapeutic tool that can combat the above limitations and serve as a supportive therapy in the management of liver diseases. Cell therapy using human fetal liver-derived stem cells can provide great potential to conservatively manage end-stage liver diseases. Therefore, the present investigation aimed to study and prove the safety and efficacy of human fetal liver-derived stem cell transplantation in patients with end-stage liver cirrhosis. Twenty-five patients with liver cirrhosis of different etiologies were infused with human fetal liver-derived stem cells (EpCAM+ve) labeled with Tc-HMPAO through hepatic artery. Our high throughput analysis using flow cytometry, RT-PCR, and cellular characterization exemplifies fetal liver cells with their high proliferation rate could be the best source for rejuvenating the diseased liver. Further, no episodes related to hepatic encephalopathy recurred in any of the subjects following hepatic stem cell transplantation. There was marked clinical improvement observed in terms of all clinical and biochemical parameters. Further, there was decrease in mean MELD score ( p < 0.01) observed in 6 months follow-up in all patients. Therapy using human fetal liver stem/progenitor cells offers a potentially supportive modality to organ transplantation in the management of liver diseases.

Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cells

To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (x10(7)/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (x10(7)/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples.

Human fetal liver derived stem cell transplantation as supportive modality in the management of end stage decompensated liver cirrhosis

Liver transplantation is the only existing modality for treating decompensated liver cirrhosis. Several factors, such as nonavailability of donors, combined with operative risks, complications associated with rejection, usage of immunosuppressive agents, and cost intensiveness, make this strategy available to only a few people. With a tremendous upsurge in the mortality rate of patients with liver disorders worldwide, there is a need to search for an alternative therapeutic tool that can combat the above limitations and serve as a supportive therapy in the management of liver diseases. Cell therapy using human fetal liver-derived stem cells can provide great potential to conservatively manage end-stage liver diseases. Therefore, the present investigation aimed to study and prove the safety and efficacy of human fetal liver-derived stem cell transplantation in patients with end-stage liver cirrhosis. Twenty-five patients with liver cirrhosis of different etiologies were infused with human fetal liver-derived stem cells (EpCAM+ve) labeled with Tc-HMPAO through hepatic artery. Our high throughput analysis using flow cytometry, RT-PCR, and cellular characterization exemplifies fetal liver cells with their high proliferation rate could be the best source for rejuvenating the diseased liver. Further, no episodes related to hepatic encephalopathy recurred in any of the subjects following hepatic stem cell transplantation. There was marked clinical improvement observed in terms of all clinical and biochemical parameters. Further, there was decrease in mean MELD score (p < 0.01) observed in 6 months follow-up in all patients. Therapy using human fetal liver stem/progenitor cells offers a potentially supportive modality to organ transplantation in the management of liver diseases.

Application of fetal neural stem cells transplantation in delaying denervated muscle atrophy in rats with peripheral nerve injury

Injury to peripheral nerves always results in progressive skeletal muscle atrophy and poor functional recovery. Previous studies have demonstrated that transplanting neural stem cells (NSCs) into peripheral nerve can differentiate into neurons and delay muscle atrophy. However, the mechanism was not very clear. In this study, we transplanted the fetal NSCs to the injured nerve and examined new formed neuromuscular junctions (NMJs) in the denervated muscle and arrest of muscle atrophy. In our study, two pregnant Fischer rats were used to harvest fetal NSCs, 70 rats were randomly divided into NSC-transplanted and control groups, five rats without surgery were used as the normal control. A volume of 5 microl culture media with or without fetal NSCs (5 x 10(6)) were transplanted into distal tibial nerve stump after the nerve was transected in two groups, respectively. Three, five, and seven months after denervation, the dry weight of gastrocnemius muscle was found significant heavier, and the fiber area was more retained in NSC-transplanted group comparing to the control group (P < 0.05). Neurons were found in the distal tibial nerves even 7 months after fetal NSCs transplantation. Newly formed NMJs were detected by immunohistochemistry. In addition, the results of electrophysiological analysis and retrograde tracing manifested that the neural pathway between muscle and differentiated neurons was integrity. In conclusions, our study demonstrated that fetal NSCs transplanted into peripheral nerves could differentiate into neurons and form functional NMJs with denervated muscle, which may be beneficial for the treatment of muscle atrophy after peripheral nerve injury.

The Maternal Immune Response to in Utero Hematopoietic Stem Cell Transplantation.

Abstract 64In utero hematopoietic stem cell transplantation (IUHSCTx) is a promising treatment strategy for many congenital hematopoietic disorders such as immunodeficiencies. However, clinical applications have been hampered by lack of engraftment, possibly secondary to a host immune response. This has been a conundrum in the field, since the fetus can also be tolerized to allogeneic cells in some circumstances. We hypothesized that it is the maternal immune response which limits engraftment of in utero transplanted cells. Methods:Fetal BALB/c mice at 14 days' gestation were transplanted with age-matched fetal liver (FL) cells (2.5 × 106 cells/fetus) from allogeneic C57B6 mice and levels of circulating donor cell chimerism were determined serially starting at 4 weeks after in utero transplantation. Rates of engraftment (number of chimeric pups/number of surviving pups) and levels of chimerism (donor CD45 cells/total CD45 cells) were compared to controls in which animals were transplanted with congenic cells (C57B6 (CD45.2) fetal hosts transplanted with C57B6 (CD45.1) FL). In order to determine the role of the maternal adaptive immune system, immunodeficient BALB/c.Rag−/− mothers (deficient in T and B cells) were bred to wild type BALB/c males, such that the fetuses (BALB/c.Rag+/−) would be immunocompetent. These fetuses were transplanted with C57B6 FL and rates of engraftment and levels of chimerism in these transplants were compared to those in wild type allogeneic transplants. In order to determine whether the maternal influence is caused by maternal lymphocytes trafficking into the fetus, C57B6 (CD45.2) females were bred to C57B6 (CD45.1) males, such that the fetal cells (CD45.1+/CD45.2+) could be distinguished from maternal cells (CD45.1−/CD45.2+). Fetal blood and tissues were examined for the presence of maternal cells by flow cytometry at various gestational ages. Results:The rate of engraftment after IUHSCTx in control animals transplanted with congenic cells was 14/16 (88%) and average levels of chimerism were 9.9±8.4%. In contrast, the rate of engraftment in wild-type BALB/c fetuses transplanted with allogeneic B6 cells was 11/25 (44%; p<0.05 compared to congenic), and levels of chimerism were 21±19 (p=NS), confirming there is an adaptive immune response to fetal stem cell transplantation. As expected, chimeric animals were tolerant to the donor strain by mixed lymphocyte reaction while injected, non-chimeric animals were sensitized. However, in the absence of a maternal adaptive immune system, rates of chimerism (in immunocompetent BALB/c.Rag+/− pups) increased to 100% (n=10, p<0.05 compared to wild type allogeneic) and levels of chimerism were significantly higher (44±18, p<0.05). Levels of chimerism in engrafted animals declined over time after allogeneic transplantation but not after congenic transplantation, indicating there is a second, late phase immune response to allogeneic cells. However, chimerism levels did not decline in the BALB/c.Rag+/− recipients, suggesting that the maternal immune system has long-lasting effects on the success of fetal transplantation, perhaps by priming the host immune system. In our analysis of maternal/fetal cellular trafficking, we detected maternal lymphocytes in the blood of midgestation fetuses (14±7% at E12.5–E14.5, n=9) which declined gradually and was undetectable after birth. Lineage analysis demonstrated that 45±15 % of maternal cells are Gr-1+ granulocytes and 21±15% are B cells. Trafficking of maternal cells into the fetus was increased following fetal manipulation (injection of PBS < injection of allogeneic HSC). Conclusions:There is an adaptive immune response which limits early engraftment after in utero transplantation of allogeneic cells and leads to a gradual decline in levels of chimerism in engrafted animals. However, in the selective absence of maternal T and B cells, all fetuses transplanted with allogeneic FL cells show long-term, multilineage engraftment and demonstrate donor-specific tolerance. These results indicate that the maternal immune system plays a significant role in the success of fetal HSC transplantation. Cellular trafficking between the mother and fetus may be a mechanism by which maternal lymphocytes encounter cells transplanted into the fetus. Our findings have clinical implications in that the success of IUHSCTx may be improved by harvesting cells from the mother or HLA-matching cells to the mother. Disclosures:No relevant conflicts of interest to declare.

Long‐Term Multilayer Adherent Network (MAN) Expansion, Maintenance, and Characterization, Chemical and Genetic Manipulation, and Transplantation of Human Fetal Forebrain Neural Stem Cells

Human neural stem/precursor cells (hNSC/hNPC) have been targeted for application in a variety of research models and as prospective candidates for cell-based therapeutic modalities in central nervous system (CNS) disorders. To this end, the successful derivation, expansion, and sustained maintenance of undifferentiated hNSC/hNPC in vitro, as artificial expandable neurogenic micro-niches, promises a diversity of applications as well as future potential for a variety of experimental paradigms modeling early human neurogenesis, neuronal migration, and neurogenetic disorders, and could also serve as a platform for small-molecule drug screening in the CNS. Furthermore, hNPC transplants provide an alternative substrate for cellular regeneration and restoration of damaged tissue in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Human somatic neural stem/progenitor cells (NSC/NPC) have been derived from a variety of cadaveric sources and proven engraftable in a cytoarchitecturally appropriate manner into the developing and adult rodent and monkey brain while maintaining both functional and migratory capabilities in pathological models of disease. In the following unit, we describe a new procedure that we have successfully employed to maintain operationally defined human somatic NSC/NPC from developing fetal, pre-term post-natal, and adult cadaveric forebrain. Specifically, we outline the detailed methodology for in vitro expansion, long-term maintenance, manipulation, and transplantation of these multipotent precursors.

Fetal Immune Responses Post In Utero Transplantation of Embryonic Stem Cells, Bone Marrow and Fetal Liver Hematopoietic Stem Cells.

Introduction: We have previously shown that in utero transplanted ES cells survive and integrate into the fetus development and have established a murine model for the study of the in vivo differentiation of ES cells. The goal of this study was to monitor the fetal immune responses post in utero transplantation of ES cells, bone marrow (BM) and fetal liver (FL) hematopoietic stem cells.Materials and Methods: Murine (MHC)-mismatched ES cells genetically engineered to express yellow fluorescent protein (YFP-ES cells) were cultured on mitomycin-treated feeder layers for four days prior to in utero transplantation (IUT) in medium containing leukemia inhibitory factor. A cell dose of 5x104 YFP-ES cells ((H-2kb) was injected intraperitoneally in the fetuses of Balb/c (H-2Kd) pregnant mice at E12- E14. BM and FL hematopoietic stem cells (H-2kb) were transplanted in utero at a dosage of 1×109 cells/kg fetal body weight into E12-E14 BALB/c fetuses (H- 2Kd). Fetal immune responses were monitored by in vitro mixed lymphocyte reaction and cytotoxicity assays performed with self (Balb/c), allogeneic ES cells, BM or FL hematopoietic cells, and 3rd party (C3H cells). Cytokine levels (IL-2, IFN-gamma, IL-4 and IL-10) were determined in the cell culture supernatants from cytotoxicity assays. Liver tissue sections were prepared from in utero transplanted fetuses and examined for the presence of lymphocytic infiltration.Results: In utero transplantation of YFP-ES-cells did not induce tolerance in the fetuses and was associated with increased cytokine production compared with BM and FL groups. Microscopic examination of liver sections of ES cell transplanted group revealed the presence of marked inflammatory infiltrate.Conclusions: Embryonic stem cells transplanted in utero induce fetal immune responses and increased cytokine production associated with (MHC) upregulation.

Functional motoneurons develop from human neural stem cell transplants in adult rats

We have shown previously that primed human fetal neural stem cells, after transplantation into rat spinal cords, differentiated into cholinergic motoneurons that sent axons to contact medial gastrocnemius myocytes. Here we demonstrate that (i) axons from the transplanted cells are cholinergic and myelinated, (ii) putative synapses form on transplanted somata and dendrites in the ventral horn, (iii) human fetal neural stem cells transplantation led to normal electromyograms from medial gastrocnemius muscles, and (iv) the gait of transplanted animals was much improved. Accumulatively, our data indicate that some transplanted human fetal neural stem cells in adult motoneuron-deficient ventral horns differentiate into relatively normal motoneurons that are integrated into spinal and peripheral circuitry. These findings are steps towards the long-term goal of providing stem cell transplants for motoneuron loss.

Role of Fetal Stem Cells in Maternal Tissue Regeneration

Microchimerism refers to the status of harboring cells from another individual at low levels. It is well known that cells traffic bidirectionally between fetus and mother during pregnancy. This situation resembles a naturally occurring long lasting fetal stem cell transplantation. The fetus acts as the donor and the mother acts as the recipient. To study the role of microchimerism in tissue regeneration, we constructed a murine microchimerism model with wild type C57BL/6J female mice carrying progenies which expressed green fluorescent proteins (GFP). Our data indicated that skin injuries in the female mice during pregnancy increased microchimerism of GFP expressing cells from the GFP transgenic progenies. The GFP positive cells also appeared at the site of spinal cord where injury occurred during pregnancy. Our study suggests that the amount of fetal cells in maternal mice significantly increased if injuries occurred during pregnancy. Fetal stem cells appear to respond to maternal injury signals and may play a role in maternal tissue regeneration during pregnancy.

Pathogens in Maternal Blood and Fetal Cord Blood Using Q-Pcr Assay

To evaluate the rates of infection of asymptomatic fetuses and mothers through fetal cord blood and maternal blood examination results. Quantitative PCR (Q-PCR) was used to detect pathogens in maternal peripheral blood and fetal cord blood after delivery of term pregnancy at Buddhist Tzu Chi Medical Center, Hualien, between July 2002 and June 2003. We used Q-PCR to detect pathogens in 29 samples of maternal blood. The maternal hepatitis B virus (HBV) DNA detection rate was 51.72% (15/29); for human cytomegalovirus DNA, the detection rate was 10.34% (3/29) and for Chlamydia trachomatis DNA the detection rate was 3.45% (1/29). No Neisseria gonorrhoeae DNA was detected. Whereas, in 29 samples of paired fetal cord blood, the detection rates were 27.59% (8/29), 10.34% (3/29), and 3.45% (1/29) for HBV DNA, C. trachomatis DNA, and N. gonorrhoeae DNA, respectively. No human cytomegalovirus DNA was detected in fetal cord blood. Our results revealed an unexpectedly high incidence of pathogens in fetal cord blood. Screening for the above pathogens in donor cord blood in cord blood banks using Q-PCR is strongly urged to decrease morbidity and mortality rates in fetal cord blood stem cell transplant recipients.

Terapia celular para acidente vascular cerebral isquêmico: esperança ou panacéia?

. These cells, which are present in variable proportions in the adult organs, could theoretically generate any living tissue of a particular organism. Although the concept of (fetal) stem cell transplantation is not new, considerable interest has arisen since the demonstration of such cells in the adult bone marrow, which could potentially provide enough cells for autologous transplantation

D-4F Restores eNOS-Dependent Vasodilation in Sickle Cell Disease by Decreasing Proinflammatory HDL.

Background: Vasoregulation is impaired in human and murine sickle cell disease (SCD). Chronic inflammation and oxidative stress impair vasodilation. High-density lipoprotein (HDL) plays an important role in attenuating inflammatory responses. Previously we showed 4F, an apoA-I mimetic designed to improve HDL function, dramatically restores vasodilation in SCD mice. Here, we examine mechanisms by which D-4F restores vasodilation in SCD mice and in mice made to develop SCD via fetal liver hematopoietic stem cell transplantation (HSCT). Effects of proinflammatory lipids and D-4F were determined in HSCT-SCD- LDL receptor null (Ldlr−/−) mice fed either chow or western diet (WD). The role of HDL was examined in HSCT-SCD-apoA-I null (apoA-I−/−) mice. Finally, the role of eNOS was examined in HSCT-SCD-eNOS deficient (eNOS−/−) mice. Mice were treated with or without D-4F (1mg/kg/d for 6–8 wks).Results: Total cholesterol concentrations in HSCT-SCD-Ldlr−/− mice fed lab chow were slightly increased compared to transgenic SCD mice (40–60 vs. 90–130 mg/dL, p<0.05) with no change in HDL. Acetylcholine-mediated vasodilation (Ach, 10-7 to 10-4M) in HSCT-SCD-Ldlr−/− mice was impaired compared to untreated non-SCD Ldlr−/− mice (10 vs 43%, p<0.05). D-4F restored eNOS-dependent vasodilation in HSCT-SCD-Ldlr−/− mice to the level in non-SCD Ldlr−/− mice. D-4F did not alter total cholesterol or HDL in HSCT-SCD-Ldlr−/− mice but did decrease proinflammatory HDL (580 vs 380, p<0.05), an index of oxidizability. In contrast to HSCT-SCD-Ldlr−/− mice fed chow diet, HSCT-SCD-Ldlr−/− mice fed WD had little to no ACh vasodilation (0–3%). D-4F increased vasodilation slightly in HSCT-SCD-Ldlr−/− fed WD (~12%). Total cholesterol and HDL increased in response to WD in HSCT-SCD-Ldlr−/− mice (p<0.01). D-4F induced minimal changes in total cholesterol, HDL or proinflammatory HDL in these mice. To examine the role of HDL, we found that vasodilation in HSCT-SCD-apoA-I−/− mice was reduced to ~25% compared to 65% in C57BL/6 mice (p<0.01). D-4F nearly doubled vasodilation to ~43% in HSCT-SCD-apoA-I−/− mice (p<0.05). L-NAME (100μM) blocked vasodilation in all HSCT-SCD-apoA-I−/− mice, indicating vasodilation was mediated exclusively by eNOS. In contrast, when we examined the effect of eNOS deficiency, ACh induced minimal increases in vasodilation (~22%). Dissection of cellular mechanisms mediating vasodilation revealed that a small portion HSCT-SCD-eNOS−/− mice was inhibited by L-NAME (i.e., NOS, ~12%), with none mediated by COX-prostacyclin (0%) and a small portion mediated by cytochrome P450 (~10%). Inhibitor studies revealed D-4F restored vasodilation in HSCT-SCD-eNOS−/− mice to ~52% (p<0.05) by predominately a L-NAME-inhibitable mechanism (NOS = 40%; COX-prostacylcin = 0% and cytochrome P450 = 11%).Conclusions: D-4F improves eNOS-dependent vasodilation even when hypercholesterolemia is superimposed on SCD. Measurements of proinflammatory HDL reveal D-4F restores vasodilation by protecting HDL against oxidation. Interestingly, D-4F protects vasodilation even in mice that have low levels of apoA-I-deficient HDL. Taken together, these data indicate proinflammatory HDL plays a critical role in mechanisms by which SCD impairs eNOS-dependent vasodilation and D-4F increases vasodilation, at least in part, by decreasing proinflammatory HDL in SCD.

Engraftment Kinetics of Human Cord Blood and Murine Fetal Liver Stem Cells Following In Utero Transplantation into Immunodeficient Mice

This study was undertaken to evaluate the kinetics of engraftment after in utero transplantation of murine fetal liver and human cord blood stem cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. NOD/SCID fetuses were injected with murine fetal liver or human cord blood CD34+ cells at day 13.5 of gestation. Frequencies of donor cells were analyzed by flow cytometry up to 48 h post transplantation and 4-16 weeks postnatally. Hematopoietic multilineage reconstitution capacity was assessed. Both types of donor cells home rapidly. However, the frequency of human cord blood stem cells rapidly diminished while the murine fetal liver stem cells expanded over time, resulting in multilineage hematopoietic reconstitution. Differences in long-term reconstitution of allogeneic versus xenogeneic donor cells were ascribed to the inability of the human cells to self-renew and differentiate in the fetal mouse environment, demonstrating the limitations of this commonly used xenograph.

In utero transplantation of autologous and allogeneic fetal liver stem cells in ovine fetuses

The purpose of this study was to assess the feasibility of autologous stem cell transplantation in fetal sheep and to compare short-term engraftment of allogeneic and autologous fetal liver stem cells in an immunocompetent large animal model. Fetal liver stem cells were collected from preimmune sheep fetuses with an open or ultrasound-guided technique. After being labeled with PKH26, the cells were transplanted intraperitoneally into allogeneic and autologous fetal recipients at 48 to 64 days of gestation. Engraftment was determined by flow cytometry and real-time polymerase chain reaction 1 to 2 weeks after transplantation. Fetal loss rate was 29% (allogeneic transplantation) and 73% (autologous transplantation). Engraftment of donor cells was found in all fetuses, with a level of < or =4.7% in fetal liver, spleen, bone marrow, blood and thymus. Overall, there was no difference between allogeneic and autologous grafts. Autologous in utero transplantation of fetal liver stem cells in fetal sheep is feasible, but yields a high loss rate. Differences in the major histocompatibility complex between donor and recipient seems not to have a major impact on stem cell engraftment early in gestation; major histocompatibility complex-independent donor/host competition might be responsible for low engraftment in immunocompetent recipients.