Progress 07/01/03 to 06/30/09
Outputs
OUTPUTS: Mentoring students Meeting presentations PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We re-established a line of sheep that accurately recapitulates the genetics, inhibitor formation, and clinical symptomatology of the severe form of human HA. Like humans, sheep sequester a large percentage of the FVIII carrier protein, vWF, within their platelets, and immune development in sheep closely parallels that in human. Collectively, these characteristics make this model ideal for studying not only new therapies for HA, but also the immunological aspects of HA and the mechanisms of FVIII inhibitor formation.
Publications
- Porada C.D., Sanada C., Long C.R., Wood J.A., Desai J., Bormann C., Menges S.L., Hanna C., Flores-Foxworth G., Shin T., Westhusin M.E., Liu W., Glimp H., Zanjani E.D., Lozier J.N., Pliska V., Stranzinger G., Joerg H., Kraemer D.C., Almeida-Porada G. Clinical and molecular characterization of a re-established line of sheep exhibiting hemophilia A. Thrombosis and Hemostasis Almeida-Porada G., Porada, C.D., Torabi A., Zanjani, E.D. Formation of human hepatocytes by human hematopoietic stem cells in sheep. Blood 104(8):2582-90, 2004. Chamberlain J, Yamagami T, Colletti E, Theise ND, Desai J, Frias A, Pixley J, Zanjani ED, Porada CD, Almeida-Porada G. Efficient generation of human hepatocytes by the intrahepatic delivery of clonal human mesenchymal stem cells in fetal sheep. Hepatology. 46(6):1935-45, 2007 \Colletti E., Airey J.A., Liu W., Simmons P.J., Zanjani E.D., Porada C.D., Almeida-Porada G. Generation of tissue-specific cells by MSC does not require fusion or donor to host mitochondrial/membrane transfer. Stem Cell Research 2(2):125-38, 2009 Almeida-Porada G., Desai J., Long C., Westhusin M., Pliska V., Stranzinger G., Joerg H., Thain D., Glimp H., Kraemer D., Porada C.D. Re-establishment and characterization of an extinct line of sheep with a spontaneous bleeding disorder that closely recapitulates human hemophilia A. Blood 110(11):347a, 2007 Sanada C., Wood J.A., Liu W., Lozier J.N., Almeida-Porada G., Porada, C.D. A Frame Shift-Induced Stop Codon Causes Hemophilia A in Sheep. Blood 112: 3378a, 2008. Bormann C, Long C, Menges S, Hanna C, Foxworth G, Shin T, Westhusin M, Pliska V, Stranzinger G Joerg, H, Glimp H, Millsap L, Porada C., Almeida-Porada G. and D. Kraemer Re-establishment of an extinct strain of sheep from a limited supply of frozen semen. Reproduction, Fertility and Development. 18:193 2006 Bormann, C., Long, C., Menges, S., Hanna, C., Foxworth, G., Westhusin, M., Pliska, V., Stranzinger, G., Glimp, H., Millsap, L., Porada, C., Almeida-Porada, G., Kraemer, D. , Reestablishment of an extinct strain of sheep utilizing assisted reproductive technologies. Reproduction Fertility and Development 2007. 21(1): p. 153. Shin T., Long C., Foxworth G., Hanna C., Menges S., Bormann C., Almeida-Porada G., Porada C., Glimp H., Millsap L., Westhusin M. and Kraemer D. A simple modified ICSI technique resulting in high pregnancy rates in merino sheep (Ovis Aries). Society for the Study of Reproduction, 38th Annual Meeting, Quebec, 2005.
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: We obtained 20 females that when compared to pooled control sheep plasma, exhibited slightly increased PTT levels, normal PT and platelet number, and slightly decreased FVIII:C Levels of Fibrinogen, FIX, vWF activity and vWF:ag were also normal. A second round of reproductive manipulations using the carriers'oocytes and the affected semen produced 23 more animals, 16 of which were obligate carriers with a similar phenotype. The other 8 animals exhibited prolonged bleeding from the umbilical cord that promptly stopped upon administration of purified human FVIII concentrate using recommended dosing. PARTICIPANTS: Sanada C., Wood J.A., Porada, C.D Sanada and Wood are both graduate students TARGET AUDIENCES: Hematologists and reserachers working on Hemophilia A PROJECT MODIFICATIONS: None
Impacts
Since sequence data is not yet publically available for the portion of the sheep genome containing fVIII, we began by performing reverse transcription of mRNA from spleen of normal sheep. Given the large size of fVIII mRNA, we could not obtain full length clones of the intact mRNA using only oligo-dT, so we elected to run two sets of reverse transcriptions, one using oligo-dT as a primer, and the other using a primer located within a small region of exon 14 for which sequence data was available. This approach yielded two cDNA clones, each of which represented roughly half of the fVIII mRNA. These clones were then TOPO cloned and PCR amplified using primers to the 5'-untranslated region (using sequence data available for the bovine genome) and the end of exon 14 for one clone, and primers to the beginning of exon 14 and the end of exon 26 for the other clone. These PCR products were then subjected to repeated sequencing reactions, with each reaction providing roughly 800bp of sequence from the 3'-end and 800bp from the 5-end. This data was then used to design new primers to perform subsequent sequencing, in effect walking along the mRNA until we obtained overlapping sequences. Once we had obtained the full-length sequence, we then repeated this approach using mRNA isolated from the spleen of one of the hemophiliac sheep. Our analyses revealed several conservative point mutations in the hemophiliac and identified a region of 11bp in exon 14 that differed between the wild type and the hemophiliac. Importantly, this difference included a frame-shift in the hemophiliac, which introduced a stop codon. Thus, the hemophilia A in these sheep is the result of a frame shift mutation in exon 14 that introduces a premature stop codon, thus preventing translation of the full length fVIII mRNA.
Publications
- Sanada C., Wood J.A., Liu W., Lozier J.N., Almeida-Porada G., Porada, C.D. A Frame Shift-Induced Stop Codon Causes Hemophilia A in Sheep. Blood 112: 3378, 2008.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This year we focused our studies on examining the ability of different populations of stem cells to give rise to/differentiate into cells able to produce FVIII by using a human-specific ELISA for detection of FVIII in plasma, immunohistochemistry staining specific for human FVIII in paraffin-embedded tissue sections, and in situ hybridization specific for human FVIIIc mRNA. Using the first method, we were able to detect human FVIII in the serum of 12 animals transplanted with human MSCs, and these results correlated with those obtained by immunohistochemistry. Although it has been reported by several groups that FVIIIc is found in human hepatocytes, we found significant levels of FVIII protein localized in hepatic sinusoidal cells. In an effort to start characterizing the mutation responsible for the observed hemophilia A within this line of sheep, we worked backwards from what Backfisch and colleagues originally described. From these early studies, we knew that the
diagnostic RFLP shows up as a result of digesting the sheep DNA with MspI, with the healthy/normal sheep presenting with a 4.2kb band and the hemophiliacs exhibiting a 5.7kb band. Armed with this knowledge, we downloaded the sequence for the entire cow X chromosome, since the X chromosome from sheep has not been sequenced and, for the most part, the homology between cow and sheep is better than 90%. While the above studies were ongoing, we used a variety of reproductive technologies to successfully obtain 20 female carriers of hemophilia A who have increased PTT levels . Although no bleeding symptomology was observed in these carriers, all had decreased levels of FVIIIc in their plasma when compared to a pooled plasma sample from control sheep Having successfully established a pool of carriers, we performed IVF using eggs from these carriers and sperm from the original hemophilic donor male and obtained 9 severely hemophilic lambs. Of these, 2 died at birth from severe bleeding, and 7
are alive and have received FVIII replacement therapy. 5 of these animals were transplanted in utero with liver, brain or BM-derived MSC. One of the transplanted animals that needed FVIII replacement therapy at birth and had multiple hemarthroses during the first 2 months of life is now free of symptoms and the levels of FVIIIc have reached normal levels, suggesting that our in utero transplant was curative. Although the results obtained with this animal are promising, we are awaiting long term outcome of the other 6 animals in order to draw a definite conclusion regarding the overall efficacy of in utero stem cell transplantation as a therapy for hemophilia A.
PARTICIPANTS: Graca Almeida-Porada,As the Principal Investigator of this proposal she will be responsible for the overall execution of the proposed research in cell therapy She will perform the required in utero stem cell transplant surgeries and be responsible for overseeing all sample analysis and interpreting results Christopher Porada As a Co-PI he will assist the PI with all the in utero stem cell transplant surgeries and use his experience with molecular biology for RFLP analysis of the hemophilic sheep and carriers He will assist in the design, execution, and interpretation of the studies involving transduction of stem cells with Factor VIII vectors, if needed Dr Kraemer, from Texas A&M University will provide coordination of the embryo tranfer procedures and will assist with the surgical and laparoscopic procedures for oocyte collection and embryo transfer Dr Westhusin will supervise the vitro fertilization and nucleus transfer activities The Veterinary Surgeon will be the
lead surgeon for the laparoscopic and surgical procedures
TARGET AUDIENCES: Hemophiliac patients and families. Scientists Hematologists
Impacts
We were able to re-establish a strain of sheep that exhibit spontaneous factor VIII deficiency with symptomology closely mimicking that of human hemophilia A. This large animal model will open new opportunities for not only the study of stem cell transplantation to cure hemophilia A, but can also be used in the future to study gene therapy approaches to treat this disease. We have also found means of augmenting hepatocyte production upon in-utero transplantation by switching the route from intra-peritoneal to intra-hepatic. By characterizing the cells within the liver producing FVIII we may be able to use different sources of stem cells to specifically generate the cells that are producing FVIII
Publications
- Almeida-Porada, M. G., Porada, C. D. (2007). Re-establishment and characterization of an extinct line of sheep with a spontaneous bleeding disorder that closely recapitulates human hemophilia A. (111st ed., vol. 110, pp. 347a). Blood.
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Progress 01/01/06 to 12/31/06
Outputs
Thus far we have establish a line of sheep female carriers of hemophilia A, that upon breeding with normal rams will produce offspring in which 0.25 of the males will be hemophiliac and 0.25 of the females will be carriers. By employing normal reproduction, we will be able to preserve a pool of hemophiliac A carriers to allow short- and long-term studies using this model. However, in order to accelerate the process of transplanting hemophiliac males fetuses with stem cells, 1 carrier ewe was artificiality inseminated using the frozen-thawed semen from the original hemophiliac sheep, and a normal ewe was made a surrogate mother of an embryo created by in vitro fertilization using a carrier egg and the sperm of the hemophiliac ram. These hemophiliac lambs were transplanted in utero at 60 days of gestation with human mesenchymal stem cells and are due to be born on May-5-10th. Characterization of the female carriers was also performed this last year by evaluation of PTT
and F8C levels. All of the ewes had increased PTT levels and decreased levels of F8C in their plasma when compared to a pooled plasma sample of control sheep. PTT mean levels of the carrier hemophiliac ewes was 82.17 and FVIII:C levels were 21.2, attesting to their status as hemophilia A carriers. To genetically confirm the hemophiliac carrier status of the females produced from the frozen/thawed semen from the hemophiliac male, we are performing an RFLP analysis as previously described by Backfisch et al. In short, we generated a FVIII-specific probe using the plasmid pF8.1 (pUC12 containing the entire 9kb human FVIII cDNA, kindly provided by Dr. Jane Gitschier at UCSF). This plasmid was double-digested with Sst I (Sac I) and Sal I, liberating 3 fragments; a 2.7kb vector fragment, and 3kb and 6kb FVIII fragments. The 3kb fragment was extracted from gel and re-digested with Kpn I enzyme. This digest produced 2 fragments; a 1.2kb and a 1.8kb. The 1.8kb fragment was then cut from the
gel and labeled with digoxigenin using the Dig-High-Prime labeling system (Roche), and subsequently used as the probe for the RFLP analysis on the carrier ewes. To perform the RFLP analysis, genomic DNA was obtained from the peripheral blood of all of the suspected carriers and from an age-matched control ewe. 10ug of each DNA was digested with Msp I, based on the findings of Backfisch et al. These digested DNA samples were then run on an agarose gel, transferred to GeneScreen Plus membrane (DuPont), probed with the digoxigenin-labeled 1.8kb human FVIII probe, and detected colorimetrically using a commercially available kit (Dig-High-Prime Labeling and Detection Kit from Roche). This analysis reveals an RFLP in hemophiliac carrier ewes, with two allelic bands presenting at 5.8kb (hemophiliac) and 4.2kb (normal). Using this system, normal ewes possess only the 4.2kb allele, and hemophiliacs possess only the 5.8kb allele.
Impacts
We were able to re-establish a strain of sheep that exhibited spontaneous factor VIII deficiency with symptomology closely mimicking that of human hemophilia A. This large animal model will open new opportunities for not only the study of stem cell transplantation to cure hemophilia A, but can also be used in the future to study gene therapy approaches to treat this disease. We have also found means of augmenting hepatocyte production upon in-utero transplantation by switching the route from intra-peritoneal to intra-hepatic. By characterizing the cells within the liver producing FVIII we may be able to use different sources of stem cells to generate specifically the cells that are producing FVIII
Publications
- Almeida-Porada, M. G., Porada, C. D., Zanjani, E. D. 2006 A large animal model for the study of human stem cell plasticity.. In A. Ho, E.D. Zanjani (Ed.), Frontiers in Stem Cell Transplantation.
- Yamagami, T., Porada, C. D., Chamberlain, J., Zanjani, E. D., Almeida-Porada, M. G. 2006 Alterations in host immunity following in utero transplantation of human mesenchymal stem cells (MSC). (9, Suppl. 1, , vol. 34 , pp. 39). Experimental Hematology.
- Mazhari, S., Porada, C. D., Chamberlain, J., Zanjani, E. D., Almeida-Porada, M. G. Characterization of membrane proteins of mesenchymal stem cells from human liver. 2006 (9, Suppl. 1, vol. 34, pp. 80). Experimental Hematology.
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Progress 01/01/05 to 12/31/05
Outputs
We were able to re-establish a strain of sheep that exhibited spontaneous factor VIII deficiency with symptomology closely mimicking that of human hemophilia A. This large animal model will open new opportunities for not only the study of stem cell transplantation to cure hemophilia A, but can also be used in the future to study gene therapy approaches to treat this disease. We have also found means of augmenting hepatocyte production upon in-utero transplantation by switching the route from intra-peritoneal to intra-hepatic. If more donor cells are produced in the transplanted animal, we postulate that higher levels of FVIII should be produced.
Impacts
We will continue to follow the studies as proposed in the application. We will continue our experiments to generate more female carriers of hemophilia A lambs, since we will need the hemophilia model to verify the clinical improvement that we think we can obtain by transplanting HSC into the fetus. As soon as the females are of age, we will start breeding them in order to obtain Hemophiliac sheep that we can transplant in utero. We also plan to establish somatic cell lines from affected animals that can be used in cloning and thus have a means to produce affected males more quickly and directly. We will keep working on means of optimizing in utero transplantation to produce cells capable of producing FVIII at therapeutic levels.
Publications
- Almeida-Porada G, Crapnell K, Porada C, Benoit B, Nakauchi H, Quesenberry P, Zanjani ED. In vivo haematopoietic potential of human neural stem cells. Br J Haematol. 2005 130:276-83.
- da Silva CL, Goncalves R, Crapnell KB, Cabral JM, Zanjani ED, Almeida-Porada G. A human stromal-based serum-free culture system supports the ex vivo expansion/maintenance of bone marrow and cord blood hematopoietic stem/progenitor cells. Exp Hematol. 2005 33:828-35.
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Progress 01/01/04 to 12/31/04
Outputs
The goal of first year was 1) to utilize frozen semen from a male and frozen fibroblast cells from a female to re-establish a strain of sheep that exhibited spontaneous factor VIII deficiency with symptomology closely mimicking that of human hemophilia A; and 2) use the human-sheep xenograft model of in utero transplantation to compare highly-defined populations of purified adult human bone marrow HSC and MSC for their ability to produce hepatocytes and hepatic endothelium in normal fetal sheep recipients. We also aimed to establish a time-course for the formation of hepatocytes after HSC transplantation, evaluating which HSC sources would more readily/rapidly differentiate into hepatocytes by examining the kinetics of hepatocyte formation for each of the transplanted stem cell populations. The recent changes and increased security in policies for the importation of biological products into the US led the USDA to return the cryopreserved hemophiliac ovine semen from
Switzerland, after its arrival into the US. This semen is absolutely essential to re-establish the strain of Alpine White sheep that exhibited spontaneous factor VIII deficiency. After extensive contact with the USDA, this entity felt that investigations for Bovine Spongiform Encephalopathy had priority over any other requests made to the USDA, and thus, they could not take the time to process our request of semen importation. Eventually, we were able to overcome all the problems, and we finally received the cryopreserved semen on March 12 of 2004. We are now ready to start the studies proposed in aim 1. To this end, we have begun inducing sheep to superovulate, so that we will have 12 donor ewes ready for the in vitro fertilization.
Impacts
In the present proposal, we will re-establish a line of Swiss white alpine sheep that possess a genetic lesion that produces a deficiency of factor VIII which closely parallels the human disease hemophilia A, and use this line as a pre-clinical model system to test the ability of in utero human stem cell transplantation to generate sufficient numbers of functional hepatic cells to correct the deficiency of factor VIII, hopefully providing a cure for hemophilia A.
Publications
- Almeida-Porada G, Porada C, Chamberlain J. Torabi A, Zanjani ED. Robust formation of human hepatocytes by human hematopoietic stem cells in sheep. Blood 2004 Jul 1
- Almeida-Porada G, Zanjani ED. A large animal noninjury model for study of human stem cell plasticity. Blood Cells Mol Dis. 32:77-81. 2004
- Almeida-Porada G, Porada C, Zanjani ED. The Fetal Sheep: A Unique Model System for Assessing the Full Differentiative Potential of Human Stem Cells. Yonsei Medical Journal 54: 7-14 suppl. 2004.
- Almeida-Porada G, Porada C, Zanjani ED. Plasticity of human stem cells in the fetal sheep model of human stem cell transplantation. Int J Hematol. 79(1):1-6. 2004
- Kogler G, Sensken S, Airey JA, Trapp T, Muschen M, Feldhahn N, Liedtke S, Sorg RV, Fischer J, Rosenbaum C, Greschat S, Knipper A, Bender J, Degistirici O, Gao J, Caplan AI, Colletti EJ, Almeida-Porada G, Muller HW, Zanjani E, Wernet P. A new human somatic stem cell from placental cord blood with intrinsic pluripotent differentiation potential. J Exp Med. 200:123-35. 2004
- Airey JA, Almeida-Porada G, Colletti EJ, Porada CD, Chamberlain J, Movsesian M, Sutko JL, Zanjani ED. Human mesenchymal stem cells form Purkinje fibers in fetal sheep heart. Circulation. 109(11):1401-7. 2004
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Progress 01/01/03 to 12/31/03
Outputs
Successful detection of human Factor VIII within liver sections from sheep transplanted in utero with human stem cells. One of the key technical issues to be in the proposed studies is examining the ability of transplanted human BM stem cells to give rise to functional factor VIII-synthesizing liver cells is the ability to successfully detect human cells expressing human Factor VIII in a sheep background. Since submitting this proposal, we have successfully identified an antibody that fulfills all of the necessary criteria for use in the proposed studies. This antibody from QED Biosciences is fairly unique in its ability to recognize only Factor VIII and not Factor VIII-related antigen. The antibody recognizes an epitope in the N-terminal region of the 83kDa light chain of the human Factor VIII molecule. This antibody also appears to be human-specific, since it produces no background staining on liver sections from age-matched control sheep. We have now performed
immuno-histochemical analyses on paraffin-embedded sections taken from the liver of several sheep transplanted in utero with human HSC and MSC, using this antibody, and have now clearly detected human Factor VIII-producing cells in the sheep background, providing us with two important pieces of information. First, the transplanted human cells are able to trans-differentiate sufficiently to produce detectable quantities of Factor VIII, and will thus likely be able to exert a therapeutic benefit in the sheep with hemophilia, once this line of sheep has been re-established. Secondly, we have now developed and optimized the methods necessary to compare various populations of stem cells for their ability to generate Factor VIII-producing liver cells and determine what percentage of the liver needs to be donor (human) derived in order to produce a therapeutic benefit in the hemophiliac sheep. Generation of hepatocytes by transplanted bone marrow stem cells is enhanced by intrahepatic
injection. In an effort to increase the numbers of human hepatocytes formed after in utero transplantation of human HSC and MSC, we have conducted experiments comparing the intraperitoneal route we normally employ to ultrasound-guided intrahepatic injection of the stem cell populations. Thus far, we have examined 6 lambs that were injected by ultrasound-guided intrahepatic injection to 6 lambs that were injected at the same point in gestation intraperitoneally. Thus far, we have been able to significantly increase (nearly 10-fold higher levels) the numbers of donor (human) derived hepatocytes within the lobe of the liver into which the stem cell populations were injected without increasing the incidence of fetal loss. Interestingly, the remainder of the liver contains levels of donor (human) hepatocytes that did not differ significantly from the levels seen in the sheep receiving an intraperitoneal injection of human stem cells. Thus, it appears that by simply altering the route of
administration, we have found a means of significantly increasing the potential therapeutic benefit of in utero stem cell transplantation for hemophilia.
Impacts
In the present proposal, we will re-establish a line of Swiss white alpine sheep that possess a genetic lesion that produces a deficiency of factor VIII which closely parallels the human disease hemophilia A, and use this line as a pre-clinical model system to test the ability of in utero human stem cell transplantation to generate sufficient numbers of functional hepatic cells to correct the deficiency of factor VIII, hopefully providing a cure for hemophilia A.
Publications
- No publications reported this period
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