John S. Pixley

Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep

Alternative methods to whole liver transplantation require a suitable cell that can be expanded to obtain sufficient numbers required for successful transplantation while maintaining the ability to differentiate into hepatocytes. Mesenchymal stem cells (MSCs) possess several advantageous characteristics for cell‐based therapy and have been shown to be able to differentiate into hepatocytes. Thus, we investigated whether the intrahepatic delivery of human MSCs is a safe and effective method for generating human hepatocytes and whether the route of administration influences the levels of donor‐derived hepatocytes and their pattern of distribution throughout the parenchyma of the recipients liver. Human clonally derived MSCs were transplanted by an intraperitoneal (n = 6) or intrahepatic (n = 6) route into preimmune fetal sheep. The animals were analyzed 56–70 days after transplantation by immunohistochemistry, enzyme‐linked immunosorbent assay, and flow cytometry. The intrahepatic injection of human MSCs was safe and resulted in more efficient generation of hepatocytes (12.5% ± 3.5% versus 2.6% ± 0.4%). The animals that received an intrahepatic injection exhibited a widespread distribution of hepatocytes throughout the liver parenchyma, whereas an intraperitoneal injection resulted in a preferential periportal distribution of human hepatocytes that produced higher amounts of albumin. Furthermore, hepatocytes were generated from MSCs without the need to first migrate/lodge to the bone marrow and give rise to hematopoietic cells. Conclusion: Our studies provide evidence that MSCs are a valuable source of cells for liver repair and regeneration and that, by the alteration of the site of injection, the generation of hepatocytes occurs in different hepatic zones, suggesting that a combined transplantation approach may be necessary to successfully repopulate the liver with these cells. (HEPATOLOGY 2007.)

Erythropoietin Does Not Cross the Placenta into the Fetus

Erythropoiesis in the fetus is controlled by erythropoietin (Ep). To determine the role of maternal Ep in this process, we used catheterized preparations of sheep and monkey fetuses to assess the ability of Ep administered to the mother to cross the placental barrier into the fetus. Ep was injected into pregnant sheep (3,600 IU/sheep) or monkeys (800-2,000 IU/animal) as a single intravenous dose, or into sheep in intravenous doses of 2,000 IU once every 12 h for a total of 4 injections. Maternal and fetal blood samples for Ep and reticulocyte determinations were obtained before and at intervals after Ep injections. The administration of Ep resulted in significant increases in maternal circulating Ep levels in sheep and monkeys. Despite the presence of high levels of maternal Ep, however, no increase in fetal plasma Ep levels was detected. The administration of Ep to the mother caused significant increases in reticulocyte production in the mother but not the fetus; injection of Ep directly to the fetus stimulated fetal erythropoiesis. These results demonstrate that Ep does not cross the placenta into the fetus even under conditions of chronically elevated maternal Ep levels, and suggest that red cell production in the fetus is regulated by Ep produced from sites within the fetus.

The effect of hypoxia and stem cell source on haemoglobin switching.

This study investigated whether relative changes that accompany the naturally occurring shifts in haematopoietic sites during human development play a role in haemoglobin (Hb) switching or whether Hb switching is innately programmed into cells. CD34+/Lineage− haematopoietic stem/progenitor cells (HSCs) were isolated from human fetal liver (F‐LVR), cord blood (CB), and adult bone marrow (ABM), and the Hb was characterized by flow cytometry on cultures that generated enucleated red cells. All feeder layers (stroma from F‐LVR, ABM, and human fetal aorta) enhanced cell proliferation and erythropoiesis but did not affect Hb type. HSCs from CB and F‐LVR generated the same Hb profile under normoxia and hypoxia. HSCs from ABM had single‐positive HbA and double‐positive HbA and HbF cells at normoxia and almost entirely double‐positive cells at hypoxia. Further characterization of these ABM cultures was determined by following mRNA expression for the transcription factors erythroid Kruppel‐like factor (EKLF) and fetal Kruppel‐like factor (FKLF) as a function of time in cultures under hypoxia and normoxia. The erythroid‐specific isoform of 5‐amino‐levulinate synthase (ALAS2) was also expressed under hypoxic conditions. We conclude that Hb switching is affected by the environment but not all HSCs are preprogrammed to respond.

Persistent circulating human insulin in sheep transplanted in utero with human mesenchymal stem cells

OBJECTIVE To determine if mesenchymal stem cells (MSC) derived from human fetal pancreatic tissue (pMSC) would engraft and differentiate in sheep pancreas following transplantation in utero. MATERIALS AND METHODS A three-step culture system was established for generating human fetal pMSC. Sheep fetuses were transplanted during the fetal transplant receptivity period with human pMSC and evaluated for in situ and functional engraftment in their pancreas, liver, and bone marrow. RESULTS Isolation and expansion of adherent cells from the human fetal pancreas yielded a cell population with morphologic and phenotypic characteristics similar to MSC derived from bone marrow. This putative stem cell population could undergo multilineage differentiation in vitro. Three to 27 months after fetal transplantation, the pancreatic engraftment frequency (chimeric index) was 79%, while functional engraftment was noted in 50% of transplanted sheep. Hepatic and marrow engraftment and expression was noted as well. CONCLUSION We have established a procedure for isolation of human fetal pMSC that display characteristics similar to bone marrow-derived MSC. In vivo results suggest the pMSC engraft, differentiate, and secrete human insulin from the sheep pancreas.

Immune Ontogeny and Engraftment Receptivity in the Sheep Fetus

Objective: The biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. We investigated the role fetal immune ontogeny might play in fetal transplantation tolerance in sheep. Methods: Engraftmentof allogeneic and xenogeneicHSC was determined 60 days following transplantation at different time points in sheep fetal gestation. Parallel analysis of surface differentiation antigen expression on cells from lymphoid organs of timed gestational age fetal sheep was determined by flow cytometry using available reagents. Results: An engraftment window was identified after day 52 gestation lasting until day 71 (term gestation: 145 days). This period was associated with the expression of the leukocyte common antigen CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells do not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms may be operative. In concert, surface IgM-positive cells express CD45 in the thymus at day 45, with a comparable delay in the appearance of IgM/CD45 cells in the periphery until late in the engraftment window. Conclusions: These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity is due to gestational age-dependent deletional tolerance.

Transduction of long-term-engrafting human hematopoietic stem cells by retroviral vectors

Gene therapy using retroviral vectors to transfer functional exogenous genes into hematopoietic stem cells (HSCs) promises to provide a permanent cure for a wide array of both hematopoietic and nonhematopoietic disorders by virtue of the fact that retroviral vectors permanently integrate into the host cell genome and HSCs are able to self-renew and give rise to differentiated progeny throughout the life span of the patient. However, for transduction and genomic integration to occur, the target cells must undergo cell division and express the appropriate retroviral receptor, requirements that have thus far hindered attempts at inserting exogenous genes into human HSCs in vitro. In the present studies, we used the fetal sheep xenograft model of human hematopoiesis to evaluate whether human long-term engrafting HSCs could be transduced in vivo, within a fetal microenvironment. We transplanted adult human bone marrow-derived CD34(+)Lin(-) cells into preimmune fetal sheep recipients and subsequently (19 days later) administered clinical-grade murine retroviral vector supernatants to these fetal hematopoietic chimeras. Our results demonstrate that this approach successfully transduced adult human HSCs within all seven sheep that survived the procedure, and that these transduced HSCs had the ability to serially engraft primary, secondary, and tertiary fetal sheep recipients. Transgene expression persisted throughout the serial transplantation. The successful in vivo transduction of long-term engrafting human HSCs with the existing generation of murine retroviral vectors has significant implications for developing new approaches to pre- and postnatal gene therapy.

Prolonged Hematopoietic Chimerism in Normal Mice Transplanted in utero with Human Hematopoietic Stem Cells

We have previously reported prolonged hematopoietic chimerism in normal mice transplanted in utero with human fetal hematopoietic stem cells (HSC) by flow cytometry. We now further confirm the human origin of these cells by demonstrating human DNA in the marrow of one such chimeric mouse. We also examined 42 mice born after in utero transplantation with HSC enriched from human adult marrow cells. All live-born mice were treated with recombinant human growth factors. Twelve had human cells in the peripheral blood (range: 01.–2.93%). Thymic samples were positive in 3 cases. The bone marrow of 2 mice contained cells expressing human CD34 antigen. Light scatter characteristics support the presence of multilineage hematochimerism. Human IgM was present in 2 of 4 chimeric sera tested. Thus, normal mice transplanted in utero with human HSC may permit long-term engraftment and differentiation of the human HSC.

In utero transplantation: Disparate ramifications

In utero stem cell transplantation, which promises treatment for a host of genetic disorders early in gestation before disease effect stems from Ray Owens seminal observation that self-tolerance, is acquired during gestation. To date, in utero transplantation (IUT) has proved useful in characterizing the hematopoietic stem cell. Recent observations support its use as an in vivo method to further understanding of self-tolerance. Preclinical development continues for its application as a treatment for childhood hematolymphoid diseases. In addition, IUT may offer therapeutic options in the treatment of diabetes among other diseases. Thus IUT serves as a technique or system important in both a basic and applied format. This review summarizes these findings.

Comparison of Once-Daily Captopril or Enalapril in Mild Essential Hypertension

The purpose of this study was to assess the effect of a daily low dose of the angiotensin‐converting enzyme (ACE) inhibitors, captopril or enalapril, in mild essential hypertension. Nine men with seated diastolic blood pressure between 95 and 104 mm Hg on placebo participated in the study. After one month of placebo, captopril 25 mg was administered; blood pressure, heart rate, ACE activity and plasma renin activity were measured hourly for 4 hours. Each patient then received captopril 50 mg once daily for 8 weeks and similar measurements were made 24 hours post‐dose every 2 weeks. After another month of placebo, the identical protocol was repeated after enalapril 5 mg. Although blood pressure and ACE activity decreased significantly (P < 0.05) within 2–4 hours of the acute doses of each inhibitor, neither captopril or enalapril produced significant reductions 24 hours after the small daily dose. Thus, neither ACE inhibitor alone was adequate to control blood pressure in mild hypertension when given once daily during 8 weeks of treatment.

Felty’s Syndrome

SummaryFelty’s syndrome is a complication of rheumatoid arthritis whereby patients develop neutropenia of varying severity. Although the main clinical concern is the development of serious infections, often patients remain asymptomatic or continue with clinical problems related to the rheumatoid arthritis and not to the neutropenia. There is now considerable clinical experience with the use of the recombinant human haemopoietic growth factors granulocyte and granulocytemacrophage colony-stimulating factors (G-CSF and GM-CSF) in the treatment of patients with Felty’s syndrome. The only indication for the use of either growth factor for Felty’s syndrome is the onset of infectious complications, which may be recurrent and serious. In general, when this occurs, the neutropenia is severe (<108 cells/L).The mechanism(s) underlying development of the neutropenia in Felty’s syndrome is similar to that in other forms of immune-mediated neutropenia, and in general is associated with a terminal defect in neutrophil maturation. It is likely that the maturational defect is a consequence of ‘immune based’ inhibition, although we lack detailed understanding of this inhibitory process. Growth factor therapy does not relieve the defect in terminal maturation, but in general may induce a significant improvement in the peripheral white cell count. Instances where growth factor therapy does not work appear to be due to an inability to overcome the maturational defect. Thus, the level of granulopoietic inhibition mediated by the rheumatoid process varies in severity among patients.To date, treatment options for Felty’s syndrome have included disease-modifying antirheumatic drugs, corticosteroids and splenectomy. The addition of growth factor therapy is a welcome addition to these less than optimal treatment options. However, all of the above therapies fail on occasion. Moreover, the dosage and frequency of growth factors must be titrated to keep the white blood cell count <5 × 109 cells/L, since overshoot may result in complications, the most common being exacerbation of the rheumatoid arthritis. Another mechanism by which these drugs may exacerbate rheumatoid arthritis is through activation of neutrophils. The addition of disease-modifying drugs may relieve the maturational defect, improve the peripheral white cell count and minimise disease exacerbation by limiting neutrophil exposure to the administered haemopoietic growth factor. However, long term monotherapy with G-CSF has been successfully employed without requiring disease-modifying therapy.