Formation of human hepatocytes by human hematopoietic stem cells in sheep

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2582-2590 ◽  
Author(s):  
Graça Almeida-Porada ◽  
Christopher D. Porada ◽  
Jason Chamberlain ◽  
Ali Torabi ◽  
Esmail D. Zanjani
Keyword(s):  
Stem Cells ◽  
Animal Model ◽  
Hematopoietic Stem Cells ◽  
Genetic Diseases ◽  
Liver Cells ◽  
Human Hepatocytes ◽  
Large Animal Model ◽  
Biliary Duct ◽  
Large Animal ◽  
Hematopoietic Stem

Abstract We took advantage of the proliferative and permissive environment of the developing preimmune fetus to develop a noninjury large animal model in sheep, in which the transplantation of defined populations of human hematopoietic stem cells resulted in the establishment of human hematopoiesis and led to the formation of significant numbers of long-lasting, functional human liver cells, with some animals exhibiting levels as high as 20% of donor (human) hepatocytes 11 months after transplantation. A direct correlation was found between hepatocyte activity and phenotype of transplanted cells, cell dose administered, source of cells used on a cell-per-cell basis (bone marrow, cord blood, mobilized peripheral blood), and time after transplantation. Human hepatocytes generated in this model retained functional properties of normal hepatocytes, constituted hepatic functional units with the presence of human endothelial and biliary duct cells, and secreted human albumin that was detected in circulation. Transplanting populations of hematopoietic stem cells can efficiently generate significant numbers of functional hepatic cells in this noninjury large animal model and thus could be a means of ameliorating or curing genetic diseases in which a deficiency of liver cells or their products threatens the life of the fetus or newborn.

Platelet-Targeted Expression of Human BDD-FVIII Reduces Bleeding in Canine Hemophilia A.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 691-691 ◽  
Author(s):  
Lily M. Du ◽  
Timothy C. Nichols ◽  
Sandra L. Haberichter ◽  
Paula M. Jacobi ◽  
Eric S. Jensen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Animal Model ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Hemophilia A ◽  
Biologically Active ◽  
Large Animal ◽  
Lentivirus Vector ◽  
Hematopoietic Stem ◽  
One Year

Abstract Abstract 691 Introduction: The goal of our study was to develop a clinically relevant strategy for platelet-targeted gene therapy of the commonly inherited bleeding disorder, Hemophilia A. We hypothesized that adult dogs (25 kg) affected with Hemophilia A could serve as a relevant “large animal” model to test if FVIII could be synthesized and sequestered within platelets derived from lentivirus-transduced hematopoietic stem cells. This approach is novel because it should permit the regulated release of FVIII from activated platelet progeny directly at the wound site as a physiological hemostatic response to bleeding challenges. Methods: cG-CSF/cSCF mobilized peripheral blood stem cells (PBSC) were immuno-selected for CD34 antigen from an apheresis product, transduced with a lentivirus vector under the transcriptional control of platelet-specific integrin αaIIb gene promoter driving expression of human BDD-FVIII. The PBSC (3 × 10 6/kg) were then autologously transplanted into animals that were preconditioned with Bulsulfan (5-10 mg/kg i.v.). After transplant, the dogs received oral cyclosporine to maintain levels at 200–400 ng/ml for 90 days and MMF at 8mg/kg for 35 days. Results: Two transplant recipients underwent periodic testing for incorporation and expression of the FVIII transgene as well as immune tolerance and phenotypic correction of Hemophilia A. PCR analysis detected the lentivirus vector within genomic DNA isolated from circulating peripheral blood leukocytes for more than one year after transplant. Immunofluorescence confocal microscopy showed synthesis of FVIII within tissue cultured canine megakaryocytes and circulating peripheral blood platelets. Chromogenic analysis of platelets isolated from transplanted dogs demonstrated the presence of a biologically active form of FVIII (FVIII:C) at approximately 5 mU/ml/1×108 platelets from 20 weeks through greater than one year after PBSC transplant. In contrast, FVIII:C was not detected within the plasma of these animals. This result coupled with the immunomodulation drugs may help to explain why the dogs failed to develop inhibitory antibodies to human FVIII. Following successful gene transfer and engraftment, both animals showed signs of clinical improvement of Hemophilia A: one dog has had one bleed per year for 2 years (vs expected 5–6/year) and the second dog has had no bleeds for 8 months following transplantation. In addition, one dog showed significant recovery from prolonged (months) history of gastrointestinal bleeding. These data are consistent with our previous results demonstrating synthesis, trafficking and storage of FVIII within αa-granules of human megakaryoyctes in vitro and platelets of the murine “small animal” model for Hemophilia A. Conclusions: This outcome raises the possibility of developing a protocol for delivering a locally inducible secretory pool of FVIII in platelets of patients with Hemophilia A following autologous transplantation of FVIII-transduced hematopoietic stem cells. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees. Wilcox:Amgen, INC: Research Reagents, canine growth factors: cG-CSF and cSCF.

scholarly journals Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates

Blood ◽  
2016 ◽  
Vol 127 (20) ◽  
pp. 2416-2426 ◽  
Author(s):  
Christopher W. Peterson ◽  
Jianbin Wang ◽  
Krystin K. Norman ◽  
Zachary K. Norgaard ◽  
Olivier Humbert ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Nonhuman Primates ◽  
Large Animal Model ◽  
Large Animal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Specific Manner

Key Points This study is the first to show that genome-editing approaches can modify multilineage, long-term repopulating cells in a large animal model. We demonstrate that the persistence of genome-edited hematopoietic stem cells can be tracked in vivo in a mutation-specific manner.

scholarly journals Expansion of hematopoietic stem cells in the developing liver of a mouse embryo

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2284-2288 ◽  
Author(s):  
Hideo Ema ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Mouse Embryo ◽  
Bone Marrow Cells ◽  
Fetal Liver ◽  
Cell Number ◽  
Liver Cells ◽  
Single Wave ◽  
Hematopoietic Stem ◽  
Fetal Liver Cells

Abstract The activity of hematopoietic stem cells in the developing liver of a C57BL/6 mouse embryo was quantified by a competitive repopulation assay. Different doses of fetal liver cells at days 11 to 18 of gestation were transplanted into irradiated mice together with 2 × 105 adult bone marrow cells. A long-term repopulation in myeloid-, B-cell, and T-cell lineage by fetal liver cells was evaluated at 20 weeks after transplantation. At day 12 of gestation multilineage repopulating activity was first detected in the liver as 50 repopulating units (RU) per liver. The number of RU per liver increased 10-fold and 33-fold by day 14 and day 16 of gestation, and decreased thereafter, suggesting a single wave of stem cell development in the fetal liver. A limiting dilution analysis revealed that the frequency of competitive repopulating units (CRU) in fetal liver cells at day 12 of gestation was similar to that at day 16 of gestation. Because of an increase of total fetal liver cell number, the absolute number of CRU per liver from days 12 to 16 of gestation increased 38-fold. Hence, the mean activity of stem cells (MAS) that is given by RU per CRU remained constant from days 12 to 16 of gestation. From these data we conclude that hematopoietic stem cells expand in the fetal liver maintaining their level of repopulating potential.

Posttransplantation lymphoproliferative disease in miniature swine after allogeneic hematopoietic cell transplantation: similarity to human PTLD and association with a porcine gammaherpesvirus

Blood ◽  
2001 ◽  
Vol 97 (5) ◽  
pp. 1467-1473 ◽  
Author(s):  
Christene A. Huang ◽  
Yasushi Fuchimoto ◽  
Zachary L. Gleit ◽  
Thomas Ericsson ◽  
Adam Griesemer ◽  
...  
Keyword(s):  
Animal Model ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Lymphoproliferative Disease ◽  
Large Animal Model ◽  
Large Animal ◽  
Miniature Swine ◽  
Hematopoietic Stem

Posttransplantation lymphoproliferative disease (PTLD) is a major complication of current clinical transplantation regimens. The lack of a reproducible large-animal model of PTLD has limited progress in understanding the pathogenesis of and in developing therapy for this clinically important disease. This study found a high incidence of PTLD in miniature swine undergoing allogeneic hematopoietic stem cell transplantation and characterized this disease in swine. Two days before allogeneic peripheral blood stem cell transplantation, miniature swine were conditioned with thymic irradiation and in vivo T-cell depletion. Animals received cyclosporine daily beginning 1 day before transplantation and continuing for 30 to 60 days. Flow cytometry and histologic examination were performed to determine the cell type involved in lymphoproliferation. Polymerase chain reaction was developed to detect and determine the level of porcine gammaherpesvirus in involved lymph node tissue. PTLD in swine is morphologically and histologically similar to that observed in human allograft recipients. Nine of 21 animals developed a B-cell lymphoproliferation involving peripheral blood (9 of 9), tonsils, and lymph nodes (7 of 9) from 21 to 48 days after transplantation. Six of 9 animals died of PTLD and 3 of 9 recovered after reduction of immunosuppression. A novel porcine gammaherpesvirus was identified in involved tissues. Miniature swine provide a genetically defined large-animal model of PTLD with many characteristics similar to human PTLD. The availability of this reproducible large-animal model of PTLD may facilitate the development and testing of diagnostic and therapeutic approaches for prevention or treatment of PTLD in the clinical setting.

1 GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMIC STUDIES

2016 ◽  
Vol 28 (2) ◽  
pp. 130
Author(s):  
R. Sper ◽  
S. Simpson ◽  
X. Zhang ◽  
B. Collins ◽  
J. Piedrahita
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Animal Model ◽  
Regenerative Medicine ◽  
Cancer Biology ◽  
Large Animal Model ◽  
Large Animal ◽  
Fetal Fibroblasts ◽  
Transgene Transmission

Transgenic pigs are an attractive research model in the field of translational research, regenerative medicine, and stem cell therapy due to their anatomic, genetic, and physiological similarities with humans. The development of a transgenic murine model with a fusion of green fluorescent protein (GFP) to histone 2B protein (H2B, protein of nucleosome core) resulted in an easier and more convenient method for tracking cell migration and engraftment levels after transplantation as well as a way to better understand the complexity of molecular regulation within cell cycle/division, cancer biology, and chromosome dynamics. Up to now the development of a stable transgenic large animal model expressing H2B-GFP has not been described. Our objective was to develop the first transgenic porcine H2B-GFP model via CRISPR-CAS9 mediated recombination and somatic cell nuclear transfer (SCNT). Porcine fetal fibroblasts were cotransfected with CRISPR-CAS9 designed to target the 3′ untranslated region of ACTB locus and a targeting vector containing 1Kb homology arms to ACTB flanking an IRES-H2B-GFP transgene. Four days after transfection GFP cells were fluorescence activated cell sorted. Single cell colonies were generated and analysed by PCR, and heterozygous colonies were used as donor cells for SCNT. The custom designed CRISPR-CAS9 knockin system demonstrated a 2.4% knockin efficiency. From positive cells, 119 SCNT embryos were generated and transferred to a recipient gilt resulting in three positive founder boars (P1 generation). Boars show normal fertility (pregnancies obtained via AI of wild type sows). Generated P1 clones were viable and fertile with a transgene transmission rate of 55.8% (in concordance with Mendel’s law upon chi-square test with P = 0.05). Intranuclear H2B-GFP expression was confirmed via fluorescence microscopy on 8-day in vitro cultured SCNT blastocysts and a variety of tissues (heart, kidney, brain, bladder, skeletal muscle, stomach, skin, and so on) and primary cultured cells (chondrocytes, bone marrow derived, adipocyte derived, neural stem cells, and so on) from P1 cloned boars and F1 42-day fetuses and viable piglets. In addition, chromosome segregation could be easily identified during cell cycle division in in vitro cultured stem cells. Custom designed CRISPR-CAS 9 are able to drive homologous recombination in the ACTB locus in porcine fetal fibroblasts, allowing the generation of the first described viable H2B-GFP porcine model via SCNT. Generated clones and F1 generation expressed H2B-GFP ubiquitously, and transgene transmission rates were with concordance of Mendel’s law. This novel large animal model represents an improved platform for regenerative medicine and chromosome dynamic and cancer biology studies.

scholarly journals Hepatocyte Transplantation to the Liver via the Splenic Artery in a Juvenile Large Animal Model

2019 ◽  
Vol 28 (1_suppl) ◽  
pp. 14S-24S
Author(s):  
J. Siefert ◽  
K. H. Hillebrandt ◽  
S. Moosburner ◽  
P. Podrabsky ◽  
D. Geisel ◽  
...  
Keyword(s):  
Animal Model ◽  
Splenic Artery ◽  
Metabolic Diseases ◽  
Venous Blood ◽  
Human Hepatocytes ◽  
Hepatocyte Transplantation ◽  
Large Animal Model ◽  
Large Animal ◽  
Venous Blood Flow ◽  
Thromboembolic Complications

Hepatocyte transplantation (HcTx) is a promising approach for the treatment of metabolic diseases in newborns and children. The most common application route is the portal vein, which is difficult to access in the newborn. Transfemoral access to the splenic artery for HcTx has been evaluated in adults, with trials suggesting hepatocyte translocation from the spleen to the liver with a reduced risk for thromboembolic complications. Using juvenile Göttingen minipigs, we aimed to evaluate feasibility of hepatocyte transplantation by transfemoral splenic artery catheterization, while providing insight on engraftment, translocation, viability, and thromboembolic complications. Four Göttingen Minipigs weighing 5.6 kg to 12.6 kg were infused with human hepatocytes (two infusions per cycle, 1.00E08 cells per kg body weight). Immunosuppression consisted of tacrolimus and prednisolone. The animals were sacrificed directly after cell infusion ( n=2), 2 days ( n=1), or 14 days after infusion ( n=1). The splenic and portal venous blood flow was controlled via color-coded Doppler sonography. Computed tomography was performed on days 6 and 18 after the first infusion. Tissue samples were stained in search of human hepatocytes. Catheter placement was feasible in all cases without procedure-associated complications. Repetitive cell transplantations were possible without serious adverse effects associated with hepatocyte transplantation. Immunohistochemical staining has proven cell relocation to the portal venous system and liver parenchyma. However, cells were neither present in the liver nor the spleen 18 days after HcTx. Immunological analyses showed a response of the adaptive immune system to the human cells. We show that interventional cell application via the femoral artery is feasible in a juvenile large animal model of HcTx. Moreover, cells are able to pass through the spleen to relocate in the liver after splenic artery infusion. Further studies are necessary to compare this approach with umbilical or transhepatic hepatocyte administration.

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