RESUMO
Human amniotic fluid derived progenitor cells (hAFPCs) may be multipotent and can be considered a potential tool in the field of cell therapy for haemophilia B. Their capacity to express human coagulation factor IX (hFIX) after transduction and their fate after in utero transplantation is unknown. hAFPCs isolated from second trimester pregnancies were assessed for their phenotypic markers, multilineage capacity, and expression of hFIX after transduction. Their engraftment potential was analysed in a mouse model after in utero transplantation at embryonic day 12.5. Immunohistochemistry, fluorescence in situ, ELISA and PCR were used to assess post-transplant chimeras. hAFPCs expressed several pluripotent markers, including NANOG, SOX2, SSEA4 and TRA-1-60, and could differentiate into adipocytes and osteocytes. In vitro, after transduction with hFIX and EGFP cDNAs, constitutive hFIX protein expression and clotting activity were found. Engraftment was achieved in various foetal tissues after in utero transplantation. Safe engraftment without oncogenesis was confirmed, with low donor cell levels, but persistent engraftment, into different organs (liver, heart and lung) through to 12 weeks of age. Transgenic expression of circulating hFIX was detected in recipient mice for up to 12 weeks. hAFPCs can be engrafted long-term in immunocompetent mice after in utero transplantation. Thus, cell transplantation approaches using genetically engineered hAFPCs may prove valuable for the prenatal treatment for haemophilia B.
Assuntos
Líquido Amniótico/citologia , Fator IX/metabolismo , Células-Tronco/metabolismo , Adulto , Animais , Diferenciação Celular , Células Cultivadas , Fator IX/genética , Feminino , Feto/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemofilia B/terapia , Humanos , Hospedeiro Imunocomprometido , Camundongos , Gravidez , Segundo Trimestre da Gravidez , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transplante de Células-Tronco , Células-Tronco/citologia , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVE: Using fetal goats as animal models, to establish the methodology of in utero transplantation of human hematopoeitic stem cell (HSC) under B-scan ultrasonographic guidance for prenatal therapy. STUDY DESIGN: Human HSC were directly injected into the peritoneal cavities of the recipient fetal goats at 45-55 days of gestation (term: 145 days) under the guidance of B-type ultrasound scan. After birth, the peripheral blood was collected for fluorescence assisted cell sorting (FACS), quantitative real-time PCR and fluorescence in situ hybridization (FISH) to detect and analyze the presence of human cells in the recipients. RESULTS: The 32 recipients were born alive except one miscarriage. To test for the presence of human-goat chimeras, cells from 13 randomly selected transplanted goats were collected. FACS analyses showed the presence of human cells in all the transplanted goats tested. The average proportion of CD34+ cells and GPA+(glycophorin A) cells in the peripheral blood were 1.34 +/- 1.10% and 2.80 +/- 2.10%, respectively. No CD34+ or GPA+ cells were found in the non-transplanted goats tested. The results of the quantitative real-time PCR in three engraftment goats were 1.2 x 10(4), 2.9 x 10(4), and 3.2 x 10(4) copies of human GPA DNA per mug of genomic DNA. FISH experiments showed that cells containing human specific alpha-satellite DNA sequence were present in the peripheral blood of the transplanted goats. CONCLUSIONS: The method described herein is safe and reliable, with low miscarriage risk and high chimerism rate. This approach may provide a promising animal model for potential prenatal treatment.
Assuntos
Cabras/embriologia , Modelos Animais , Transplante de Células-Tronco/métodos , Ultrassonografia/métodos , Animais , Cromossomos Humanos Par 17/genética , DNA/sangue , DNA Satélite/sangue , Feminino , Citometria de Fluxo , Idade Gestacional , Glicoforinas/genética , Cabras/sangue , Humanos , Hibridização in Situ Fluorescente , Cavidade Peritoneal/embriologia , Reação em Cadeia da Polimerase , Gravidez , Quimeras de Transplante/genética , Transplante HeterólogoRESUMO
OBJECTIVE: To analyze the existence and the dynamic cell frequencies of human cells in goats transplanted in utero with human hematopoietic stem cell (hHSC) by using fluorescence in situ hybridization (FISH) technique. METHODS: Interphase FISH (IFISH) with human-specific 17-chromosome satellite DNA and/or human-specific Y-chromosome satellite DNA as probes was performed to analyze the presence and proportions of human cells in 13 transplanted goats. Samples were peripheral blood cells, bone marrow smears and liver touch imprint preparations. RESULTS: Of the 13 transplanted goats, eleven were identified to present human cells. Among them, two goats transplanted with human male HSC were found to have human male cells. The results demonstrated that these transplanted goats were human/goat HSC xenogeneic chimeras. Human cell frequencies decreased with the goat age (months), but the longest survival reached 21 months. During the detected life periods of goats, human cell frequencies in peripheral blood, bone marrow and liver tissues were less than 1@1000, but local human cell frequencies of 207.92@1000 and 392.41@1000 were detected in the liver tissues of 2 transplanted goats. CONCLUSIONS: The existence and long-term survival of human cells in transplanted goats detected by FISH indicated that goats were appropriate recipients for hHSC in utero transplantation. The lower human cell frequencies in blood and bone marrow, and the higher local human cell frequencies in liver tissues suggested that the microenvironment of goat liver tissues might favor the survival, proliferation and differentiation of human cells.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Transplante Heterólogo , Animais , Feminino , Cabras , Humanos , Hibridização in Situ Fluorescente , Masculino , Útero/cirurgiaAssuntos
Antígenos CD34/metabolismo , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Proteínas de Fusão bcr-abl/genética , Hematopoese , Animais , Sangue Fetal/citologia , Cabras , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Transplante Heterólogo , Proteínas WT1/metabolismoRESUMO
To investigate multitissue engraftment of human primitive hematopoietic cells and their differentiation in goats, human CD34+ Lin- cord blood cells transduced with a GFP vector were transplanted into fetal goats at 45-55 days of gestation. GFP+ cells were detected in hematopoietic and nonhematopoietic organs including blood, bone marrow, spleen, liver, kidney, muscle, lung, and heart of the recipient goats (1.2-36% of all cells examined). We identified human beta2 microglobulin-positive cells in multiple tissues. GFP+ cells sorted from the perfused liver of a transplant goat showed human insulin-like growth factor 1 gene sequences, indicating that the engrafted GFP+ cells were of human origin. A substantial fraction of cells engrafted in goat livers expressed the human hepatocyte-specific antigen, proliferating cell nuclear antigen, albumin, hepatocyte nuclear factor, and GFP. DNA content analysis showed no evidence for cellular fusion. Long-term engraftment of GFP+ cells could be detected in the blood of goats for up to 2 yr. Microarray analysis indicated that human genes from a variety of functional categories were expressed in chimeric livers and blood. The human/goat xenotransplant model provides a unique system to study the kinetics of hematopoietic stem cell engraftment, gene expression, and possible stem cell plasticity under noninjured conditions.