Fetal Liver Cells Transplanted in Utero Rescue the Osteopetrotic Phenotype in the oc/oc Mouse
2009; Elsevier BV; Volume: 174; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2009.080688
ISSN1525-2191
AutoresBarbara Tondelli, Harry C. Blair, Matteo M. Guerrini, Kenneth D. Patrene, Barbara Cassani, Paolo Vezzoni, Franco Lucchini,
Tópico(s)Immune Response and Inflammation
ResumoAutosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts, which differentiate from hematopoietic precursors. In half of human cases, ARO is the result of mutations in the TCIRG1 gene, which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO, other stigmata of the disease, such as secondary neurological and growth defects, are not reversed. For this reason, ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse, a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO, we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells, which include hematopoietic stem cells, into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment, and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore, oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts, which differentiate from hematopoietic precursors. In half of human cases, ARO is the result of mutations in the TCIRG1 gene, which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO, other stigmata of the disease, such as secondary neurological and growth defects, are not reversed. For this reason, ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse, a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO, we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells, which include hematopoietic stem cells, into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment, and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore, oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder. Autosomal recessive osteopetrosis (ARO) is a severe bone disease, which, in about half of the cases, is due to mutations in the gene TCIRG1 that codes for the a3 subunit of the vacuolar proton pump.1Frattini A Orchard PJ Sobacchi C Giliani S Abinun M Mattsson JP Keeling DJ Andersson AK Wallbrandt P Zecca L Notarangelo LD Vezzoni P Villa A Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.Nat Genet. 2000; 25: 343-346Crossref PubMed Scopus (563) Google Scholar The clinical picture includes growth defects, osteosclerosis, pancytopenia due to absence of the marrow cavity, and subtle cranial malformations causing hydrocephalus and compression of nerves, with secondary blindness and deafness.2Gerritsen EJ Vossen JM Fasth A Friedrich W Morgan G Padmos A Vellodi A Porras O O'Meara A Porta F Bordigoni P Cant A Hermans J Griscelli C Fischer A Bone marrow transplantation for autosomal recessive osteopetrosis. A report from the Working Party on Inborn Errors of the European Bone Marrow Transplantation Group.J Pediatr. 1994; 125: 896-902Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Unfortunately, while pancytopenia may be rescued by postnatal bone marrow transplantation, the neurological defects cannot, since they are present at birth and are not reversible.3Driessen GJ Gerritsen EJ Fischer A Fasth A Hop WC Veys P Porta F Cant A Steward CG Vossen JM Uckan D Friedrich W Long-term outcome of haematopoietic stem cell transplantation in autosomal recessive osteopetrosis: an EBMT report.Bone Marrow Transplant. 2003; 32: 657-663Crossref PubMed Scopus (149) Google Scholar In addition, good engraftment of postnatal bone marrow cells requires conditioning with irradiation or chemical agents and is strictly dependent on histocompatibility. Because of the latter, treatment may be complicated by graft versus host disease, and transplant is effective only in a portion of cases.3Driessen GJ Gerritsen EJ Fischer A Fasth A Hop WC Veys P Porta F Cant A Steward CG Vossen JM Uckan D Friedrich W Long-term outcome of haematopoietic stem cell transplantation in autosomal recessive osteopetrosis: an EBMT report.Bone Marrow Transplant. 2003; 32: 657-663Crossref PubMed Scopus (149) Google Scholar ARO therefore is a paradigm of genetic diseases needing prenatal treatment. In addition to preventing irreversible damage, in utero transplantation (IUT) of hematopoietic stem cells (HSC) does not require MHC matching, since tolerance toward grafted cells is normally acquired by exposure to donor cells during fetal life.4Flake AW Zanjani ED In utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers.Blood. 1999; 94: 2179-2191PubMed Google Scholar With these considerations in mind, we performed IUT of adult bone marrow cells in the oc/oc mouse model, which almost perfectly recapitulates the human osteopetrotic phenotype.5Scimeca JC Franchi A Trojani C Parrinello H Grosgeorge J Robert C Jaillon O Poirier C Gaudray P Carle GF The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants.Bone. 2000; 26: 207-213Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar This strain carries a naturally occurring mutation that was originally selected due to the presence of osteopetrosis. It was later shown to be due to a genomic deletion in the 5′ region of the gene (called tcirg1 or atp6i) coding for the mouse a3 subunit of the vacuolar proton pump. This deletion causes complete absence of the corresponding protein, as occurs in human ARO patients who lack this gene product. Therefore both the mouse and the human patients bear null mutations that translate into the inability to acidify the resorbing lacuna at the osteoclast/bone interface.1Frattini A Orchard PJ Sobacchi C Giliani S Abinun M Mattsson JP Keeling DJ Andersson AK Wallbrandt P Zecca L Notarangelo LD Vezzoni P Villa A Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis.Nat Genet. 2000; 25: 343-346Crossref PubMed Scopus (563) Google Scholar, 6Taranta A Migliaccio S Recchia I Caniglia M Luciani M De Rossi G Dionisi-Vici C Pinto RM Francalanci P Boldrini R Lanino E Dini G Morreale G Ralston SH Villa A Vezzoni P Del Principe D Cassiani F Palumbo G Teti A Genotype-phenotype relationship in human ATP6i-dependent autosomal recessive osteopetrosis.Am J Pathol. 2003; 162: 57-68Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Susani L Pangrazio A Sobacchi C Taranta A Mortier G Savarirayan R Villa A Orchard P Vezzoni P Albertini A Frattini A Pagani F TCIRG1-dependent recessive osteopetrosis: mutation analysis, functional identification of the splicing defects, and in vitro rescue by U1 snRNA.Hum Mutat. 2004; 24: 225-235Crossref PubMed Scopus (89) Google Scholar These genetic and biochemical identities are the basis for the similarity of their clinical picture, making the oc/oc strain a suitable model for TCIRG1-dependent human ARO. By using this mouse model, we were able to show that injection of adult bone marrow cells at day 14.5 of pregnancy allowed complete rescue of the phenotype in two out of 14 mutant mice transplanted and partial rescue in three others.8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar This result, establishing the principle that ARO can be completely rescued by a single in utero injection of unmatched HSC, prompted us to investigate whether different sources of cells can improve the degree and the percentage of cured mice. In this regard, fetal liver, which contains HSCs, was a very promising source, since it was shown in an animal model that fetal stem cells engraft much better than adult HSC cells in fetal recipients.9Taylor PA McElmurry RT Lees CJ Harrison DE Blazar BR Allogenic fetal liver cells have a distinct competitive engraftment advantage over adult bone marrow cells when infused into fetal as compared with adult severe combined immunodeficient recipients.Blood. 2002; 99: 1870-1872Crossref PubMed Scopus (51) Google Scholar, 10Harrison DE Zhong RK Jordan CT Lemischka IR Astle CM Relative to adult marrow, fetal liver repopulates nearly five times more effectively long-term than short-term.Exp Hematol. 1997; 25: 293-297PubMed Google Scholar Fetal liver cells have also been used in the same oc/oc mouse model as source of gene therapy-targeted cells for neonatal transplantation.11Johansson MK de Vries TJ Schoenmaker T Ehinger M Brun AC Fasth A Karlsson S Everts V Richter J Hematopoietic stem cell-targeted neonatal gene therapy reverses lethally progressive osteopetrosis in oc/oc mice.Blood. 2007; 109: 5178-5185Crossref PubMed Scopus (42) Google Scholar Use of fetal liver cells for IUT in one case of human severe immunodeficiency gave good results.12Touraine JL In utero transplantation of fetal liver stem cells into human fetuses.Hum Reprod. 1992; 7: 44-48Crossref PubMed Google Scholar However, the IUT procedure has not been widely used in humans so far and, according to a recent review, only 50 cases have been reported over the past 20 years. Indeed, despite the strong rationale for IUT, there have been difficulties in achieving permanent engraftment of donor cells.13Merianos D Heaton T Flake AW In utero hematopoietic stem cell transplantation: progress toward clinical application.Biol Blood Marrow Transplant. 2008; 14: 729-740Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar On the other hand, ethical use of human fetal cells should be achievable with suitable safeguards that may be a good source for the treatment of human ARO, if proven useful in animal models. Here we show that enzymatically disaggregated fetal liver cells can cure a high proportion of oc/oc mice when injected in utero. Two pairs of (C57BL/6JxB6C3Fe-a/aF1) oc/+ mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and maintained in our central animal facility in accordance with the general guidelines released by the Italian Ministry of Health. CD-1 TG(ACTB-EGFP) transgenic mice, in which the enhanced green fluorescent protein (EGFP) gene is under the control of the chicken β-actin promoter and the cytomegalovirus (CMV) enhancer, were a kind gift of Dr. Okabe.14Okabe M Ikawa M Kominami K Nakanishi T Nishimune Y 'Green mice' as a source of ubiquitous green cells.FEBS Lett. 1997; 407: 313-319Abstract Full Text Full Text PDF PubMed Scopus (2262) Google Scholar Pregnant CD-1 TG(ACTB-EGFP) females were sacrificed at 12.5 days post coitum (p.c.). The abdomen was opened and the uterus was removed. Each GFP+ embryo was dissected away from the uterus and placed in a petri dish containing sterile PBS. The yolk sac was opened and the umbilicus was removed to allow exsanguination. Each embryo was then placed in fresh PBS, and the livers were isolated from the extraneous tissue under a stereomicroscopy. In the first set of trials, the livers were mechanically disaggregated with a Gilson P1000 pipettor. In the second set of trials, the livers were enzymatically dissociated as described by Rosen et al, 2002.15Rosen ED Cornelissen I Liang Z Zollman A Casad M Roahrig J Suckow M Castellino FJ In utero transplantation of wild-type fetal liver cells rescues factor X-deficient mice from fatal neonatal bleeding diatheses.J Thromb Haemost. 2003; 1: 19-27Crossref PubMed Scopus (17) Google Scholar Briefly, the livers were incubated in 5 ml of 0.05% trypsin for 15 minutes at 37°C. The excess solution was discarded and the disaggregated cells were resuspended by gentle pipetting. The trypsin was quenched by 5 ml of cold Dulbecco's modified Eagle's medium/F12 with 10% fetal bovine serum. The cell suspension was then passed through a 100-μm filter and the filter was rinsed with 5 ml of medium. The cells were collected by centrifugation at 400 × g for 5 minutes at 4°C, and the pellet was resuspended in 5 ml of medium. The cells were filtered again to remove material larger than 40 μm and the cells were collected as before. The cells were rinsed with 10 ml of PBS with 2% of fetal bovine serum, pelleted, and resuspended in 1 ml of 2% fetal bovine serum/PBS. The viability was determined by trypan blue exclusion, and the cells were diluted, in 2% fetal bovine serum/PBS, to a concentration of 105 cells per μl. Pregnant recipient oc/+ females mated with oc/+ male mice were anesthetized with Avertin at 13.5 days p.c. Following disinfection of the abdomen with 70% ethanol, a midline incision was performed through the skin and the peritoneal wall. The uterine horns were exposed to allow the visualization of the embryos and their livers through the uterine wall. The embryos were punctured through the peritoneum in the direction of the liver with a 50-μm diameter beveled glass capillary loaded with 2 μl of cells suspension (2 × 105 cells). The uterine horns were reinserted in the peritoneal cavity and the incisions were sutured. Genomic DNA was obtained from tail biopsies and from liver.8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar Heterozygotes (oc/+), homozygotes (oc/oc) and wild-type) animals were identified by Multiplex PCR amplification with two pairs of primers, since oc/oc mice bear a 1579 bp deletion at the 5′ end of Tcirg1 gene (GenBank AF188702), spanning from intron 1 to the 5′ of exon 3.8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar The first primer pair (OCwtF: 5′-TCATGGGCTCTATGTTCCGG-3′; OCwtR: 5′-GAAGGCGCTCACGGATTCGT-3′, indicated by the gray arrows in supplemental Figure S1C at http://ajp.amjpathol.org), detecting a sequence internal to the deleted region, identifies the wild-type allele producing a 431 bp amplification product. The second pair (OCmutF: 5′-GGCCTGGCTCTTCTGAAGCC-3′; OCmutR: 5′-CCGCTGCACTTCTTCCCGCA-3′, indicated by the black arrows in supplemental Figure S1C at http://ajp.amjpathol.org), homologous to deletion-flanking regions, identifies the deleted allele by producing a 563 bp band in the mutant allele or a 2139 bp product in the wild-type allele, which is usually not observed due to its length. Therefore, the wild-type animals show a 431 bp band, the homozygous mutants show a 563 bp band, while the heterozygous animals have both (Supplemental Figure S1, A at http://ajp.amjpathol.org). PCR was performed in 25 μl total volume with 1U Taq Polymerase (GoTaq Flexi DNA Polymerase, Promega), 1 mmol/L MgCl2, 200 μmol/L dNTP, 10pmol of each oligonucleotide and 20 ng of genomic DNA. Thermocycling consisted of a denaturation step at 94°C for 3 minutes followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing at 60.5°C for 30 seconds, and extension at 72°C for 1 minute. Southern blot was performed according to the nonradioactive DIG labeling and chemioluminescent detection protocol (Roche). The probe was DIG-labeled by PCR performed on genomic DNA of a wild-type mouse (C57BL6) using the primers OCwtF and OCmutR.8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar The probe binds a 4.7 kb fragment on the wild-type allele and a 7.0 kb on the mutated allele, because of the loss of an EcoRI restriction site in intron 1 (Supplemental Figure S1C at http://ajp.amjpathol.org). Five μg of DNA from transplanted mice (tail and liver) and untreated control mice (wild-type, oc/oc, oc/+ and GFP) were digested with EcoRI enzyme and blotted for the analysis. Cell suspensions were obtained from bone marrow by flushing of femurs and tibiae. Staining of cells for FACS analysis was performed in PBS containing 3% fetal calf serum (staining buffer) with APC-Alexa Fluor® 750-conjugated c-Kit (2B8) and APC-conjugated Sca-1 (D7) monoclonal antibodies (mAbs) obtained from eBioscience (San Diego, CA). The lineage cocktail included the following PE-conjugated mAbs purchased from BD Pharmingen: B220 (RA3–6B2), TER119 (TER-119), Mac1/CD11b (M1/70) and CD3. Percentage of donor cells within specific cell subsets was determined by detection of GFP expression. Stained cells were analyzed with a FACS Canto II flow cytometer (BD Biosciences, San Jose, CA). DIVA software (BD) was used for data acquisition and analysis. Skeletons after removal of viscera and fixation in Millonig solution were scanned by dual excitation X-ray absorptiometry (Lunar PixiMus, GE Medical Systems, Madison WI), and sections of L4–5 vertebrae, tibia, and base of the skull with mandible were cut, decalcified, and processed for paraffin embedding. Six μm sections were then cut, deparaffinized, and stained with hematoxylin and eosin and photographed, as described.8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar Lumbar vertebrae or maxillae of mice, fixed in 5% formaldehyde, were used for microcomputed tomography. Microcomputed tomography was performed on a Viva CT40 instrument (Scanco Medical, Bassersdorf, Switzerland) with three-dimensional reconstruction with the instrument-specific software provided by the instrument manufacturer. The scan slice increment was 10 μm, and three dimensional reconstructions of the spine used a density cutoff of 200 mg/cm3. For analysis of the maxilla, total mineralized tissue reconstructions used a density cutoff of 450 mg/cm3, and to determine teeth and very dense osteopetrotic bone, a density cutoff of 650 mg/cm3 was used. The culture conditions were as described.8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar Briefly, bone marrow cells were flushed from femora and tibiae in IMDM with 2% FCS and cultured at 104−105 per ml in Methocult medium M03434 (StemCell Technologies) in duplicate. Cultures were scored at 2, 7 and 9 days with an inverted fluorescent microscope using standard criteria. At 12.5 days p.c., HSCs in the fetal liver are in exponential growth and at 13.5 days p.c. they start to migrate to spleen and bone marrow, where they settle and colonize.16Dzierzak E Medvinsky A Mouse embryonic hematopoiesis.Trends Genet. 1995; 11: 359-366Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 17Moore MA Commentary: the role of cell migration in the ontogeny of the lymphoid system.Stem Cells Dev. 2004; 13: 1-21Crossref PubMed Scopus (29) Google Scholar Fetal liver cells were obtained from day 12.5 p.c. fetuses heterozygous for the CMV-EGFP transgene in CD-1 background.14Okabe M Ikawa M Kominami K Nakanishi T Nishimune Y 'Green mice' as a source of ubiquitous green cells.FEBS Lett. 1997; 407: 313-319Abstract Full Text Full Text PDF PubMed Scopus (2262) Google Scholar Cells were injected in day 13.5 p.c. fetuses originated from oc/+ x oc/+ mating. Mice born from IUT were initially screened by PCR on DNA extracted from tail biopsies (see supplemental Figure S1A at http://ajp.amjpathol.org). Transplanted mice identified as oc/oc mice were analyzed by Southern blotting to confirm their genotype (see supplemental Figure S1, C–D at http://ajp.amjpathol.org). All of the rescued oc/oc mice showed only the expected mutated 7.0-kb band in their tail DNA. It is interesting to note that in the liver of mouse F4 and, to a lesser degree, of mouse F2, also a 4.7-kb band corresponding to the wild-type gene is detected. This band, derived from the transplanted GFP-positive cells, is undetectable in DNA from the tail of the same individuals and witness the high level of chimerism achieved in the liver, as determined by observation with an inverted fluorescent microscope. Two sets of trials were performed in which alternative methods for disaggregating liver tissue (mechanical or enzymatic) were used. In the first trial, 19 injected females delivered 60 pups. Among these, only one of the eight oc/oc mutant mice survived more than 4 weeks (Table 1), which is the maximum survival age for untreated oc/oc mice. This female mouse (F1) was phenotypically normal, as judged by growth (see supplemental Figure S2 at http://ajp.amjpathol.org) and behavior. At 10 days of age, incisor teeth showed a regular eruption, making the animal able to feed (see supplemental Figure S2 at http://ajp.amjpathol.org); nevertheless a malocclusion of the incisors prevents them from being worn down, requiring a periodic trimming of these teeth. This aspect of tooth growth has been reported also in other papers, which obtained significant rescue of the oc/oc phenotype by postnatal treatment.11Johansson MK de Vries TJ Schoenmaker T Ehinger M Brun AC Fasth A Karlsson S Everts V Richter J Hematopoietic stem cell-targeted neonatal gene therapy reverses lethally progressive osteopetrosis in oc/oc mice.Blood. 2007; 109: 5178-5185Crossref PubMed Scopus (42) Google Scholar, 18Johansson M Jansson L Ehinger M Fasth A Karlsson S Richter J Neonatal hematopoietic stem cell transplantation cures oc/oc mice from osteopetrosis.Exp Hematol. 2006; 34: 242-249Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The behavior of F1 appeared normal, since it explored the space around like wild-type mice. When it was sacrificed at 30 weeks of ages, it reaches the weight of 22 grams.Table 1Summary of Fetal Liver Cell in Utero Transplantation ExperimentsExperimentFemales injectedPupsOc/ocOc/oc rescued% Rescue119608112,5220459555,6Totals3910517635,3 Open table in a new tab In the second trial, 20 females were injected and 45 pups were born (Table 1 and see supplemental Figure S2 at http://ajp.amjpathol.org). Five out of nine oc/oc mutant mice survived more than 4 weeks: three females (F2, F4, and F5) and two males (M3 and M6). F5 and M3 showed tooth eruption and performed a normal life but, like F1, developed malocclusion. F5 was sacrificed at 24 weeks when it weighed about 20 grams. M3 died of unknown causes at 24 weeks when it weighed around 25 grams. F2 and F4 didn't show tooth eruption and were fed on ground pellets and corn pudding. Despite the absence of teeth, F2 reached the weight of 12 grams and F4 17 grams. M6 died at the age of 5 weeks and it showed a partial tooth eruption. F2, M3, F4, and F5 were examined in a behavior test in comparison with an untreated oc/oc mutant mouse. They showed a normal capacity of exploring the surrounding environment, being able to search food, to climb toward it, and to feed. They didn't show abnormal movement or the typical turning movement of the oc/oc mutant mice. M3 and F5 were mated with wild-type partners. M3 produced two litters of 18 mice in total and F5 delivered four mice, all of which were oc/+ (see supplemental Figure S1B at http://ajp.amjpathol.org), confirming their homozygous status and suggesting that TCIRG1-defective ARO patients have no inherent defect in germ line maturation. The difference in rescue between the two methods, one of eight oc/oc mice in mechanical disaggregation versus five of nine for enzymatic disaggregation, has a χ2 3.44, which with one degree of freedom corresponds to P = 0.06. Relative to survival of five of 14 pups using adult marrow,8Frattini A Blair HC Sacco MG Cerisoli F Faggioli F Cato EM Pangrazio A Musio A Rucci F Sobacchi C Sharrow AC Kalla SE Bruzzone MG Colombo R Magli MC Vezzoni P Villa A Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero.Proc Natl Acad Sci USA. 2005; 102: 14629-14634Crossref PubMed Scopus (55) Google Scholar the liver cells were not significantly better, but in the rescued animals the phenotypes of surviving animals were qualitatively better (see Discussion). Table 2 summarizes the characteristics of each animal born from IUT as compared with two wild-type mouse (C57BL6), male and female. It is noteworthy that some of the cured animals were characterized by splenomegaly (F1, F2, and F4). The reason why a small degree of splenomegaly is maintained in mice that showed a good rescue of the phenotype is not clear. It could be that the total volume of bone marrow cavities is still below the normal values in rescued mice or, alternatively, that extramedullary spleen sites are also occupied when, during the last days of pregnancy, the transplanted cells have not yet completely remodeled the bones. Likewise, we cannot explain the relatively incomplete rescue of the tooth phenotype, although it could be hypothesized that for some reasons osteoclast function in the jaw is not properly regulated in the treated mice (see also the findings at the microCT analysis of jaws).Table 2Clinical and Pathological Parameters of IUT-Treated oc/oc MiceMouseBody length (cm) without tailBody weight (g)Spleen weight (mg)Spleen length (cm)Age of death (weeks)Cause of deathMatedF1922, 503703, 530SacrificedNoF26, 512, 61320216SacrificedNoM3925, 00NDND24SpontaneousYesF47, 517, 18190224SacrificedNoF5820, 57801, 524SacrificedYesM6NDNDNDND5SpontaneousNoFWt821, 3040124SacrificedNDMWt1028, 01301, 524SacrificedNDThe measures of body length, body weight, spleen length, and spleen weight were taken at the age of sacrifice. ND, not determined; FWt, female wild-type; MWt, male wild-type; treated animals are numbered as in the text. Some parameters were not reported in animals which died unexpectedly. Open table in a new tab The measures of body length, body weight, spleen length, and spleen weight were taken at the age of sacrifice. ND, not determined; FWt, female wild-type; MWt, male wild-type; treated animals are numbered as in the text. Some parameters were not reported in animals which died unexpectedly. In four mice the skeletal phenotype was investigated by X-ray and compared with a female wild-type mouse of the same age (Figure 1A). There was a good correlation between the skeletal appearance, the presence of marrow cavities, the mineral density and the clinical findings. F2 female, which was the smallest of these mice, was the one showing residual signs of osteopetrosis, while F4 and F5 were essentially normal, as was the M3 mouse. In particular, normal marrow cavity was observed in the long bones from all of these three mice (Figure 1B). The animal bodies, after harvest of organs and partial disarticulation, were sent for histological analysis with the observers blinded to genotype. At this stage, dual energy X-ray absorptiometry (DEXA; Lunar PixiMus) was performed (Figure 2A), which showed only highly localized densities in some animals, consistent with treated osteopetrosis. Bone densities of F1 and F5 could not be distinguished from normal mice, and median bone densities of the animals correlated with the weight of the animals but otherwise showed no significant differences (not illustrated). Sections of the spine, tibia, and skull were also examined. This showed (Figure 2B) that most of the treated animals, including animals that were apparently completely healthy, had microscopic areas of osteopetrosis with retained mineralized cartilage and sclerotic bone, including F2; sections of F4 showed no abnormalities, but focal lesions were apparent on DEXA, particularly in the skull. The most obviously affected animals were the small male M6, with clear skull lesions on DEXA and significant osteopetrosis near the ends of long bones and vertebral
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