Array-MLPA: comprehensive detection of deletions and duplications and its application to DMD patients.

Multiplex ligation-dependent probe amplification (MLPA) is widely used to screen genes of interest for deletions and duplications. Since MLPA is usually based on size-separation of the amplification products, the maximum number of target sequences that can be screened in parallel is usually limited to approximately 40. We report the design of a robust array-based MLPA format that uses amplification products of essentially uniform size (100-120 bp) and distinguishes between them by virtue of incorporated tag sequences. We were thus able to increase probe complexity to 124, with very uniform product yields and signals that have a low coefficient of variance. The assay designed was used to screen the largest set studied so far (249 patients) of unrelated Duchenne muscular dystrophy (DMD) cases from the Chinese population. In a blind study we correctly assigned 98% of the genotypes and detected rearrangements in 181 cases (73%); i.e., 163 deletions (65%), 13 duplications (5%), and five complex rearrangements (2%). Although this value is significantly higher for Chinese patients than previously reported, it is similar to that found for other populations. The location of the rearrangements (76% in the major deletion hotspot) is also in agreement with other findings. The 96-well flow-through microarray system used in this research provides high-throughput and speed; hybridization can be completed in 5 to 30 minutes. Since array processing and data analysis are fully automated, array-MLPA should be easy to implement in a standard diagnostic laboratory. The universal array can be used to analyze any tag-modified MLPA probe set.

Derivation of human embryonic stem cell lines from parthenogenetic blastocysts.

Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting.

In utero transplantation of human hematopoietic stem/progenitor cells partially repairs injured liver in mice.

The aim of this study is to establish a novel mouse model with high achievement and chimerism by in utero transplantation of human hematopoietic stem/progenitor cells and to explore the possibility that human adult hematopoietic stem/progenitor cells can differentiate into hepatocyte-like cells and partially repair the liver damage induced by carbon tetrachloride (CCl(4)). Mononuclear cells (MNCs) were isolated from fresh human umbilical cord blood (hUCB) and CD34(+) cells were enriched from the MNCs by magnetic cell isolation. These cells were injected respectively into the fetal mice at 11-13 days of gestation. At one month after birth, the specific markers of human cells, human alpha-satellite sequence (h17alpha), CD14, CD34, CD45, and GPA were detected by PCR and FACS. At three and six months after birth, the established human-mouse chimeras were administered with CCl(4) by intraperitoneal injection. The biochemical markers (ALT, AST, ALP, albumin) in serum were determined and human hepatocyte-specific proteins, such as human albumin, hepatocyte nuclear factor-4, hepatocyte-specific antigen, tryptophan 2,3-dioxygenase and alpha fetoprotein were analyzed by PCR, RT-PCR, real-time PCR and immunohistochemistry staining, respectively. More than 77% of recipients demonstrated human-mouse chimera. Significantly, hUCB hematopoietic stem/progenitor cells may differentiate into human hepatocyte-like cells with evidence of the expression of human hepatocyte-specific proteins as well as partially repair or protect liver damage induced by CCl(4). The mouse model described in this article provides a useful tool for the studies of regeneration of human hepatocyte-like cells from adult hematopoietic stem/ progenitor cells as well as facilitates the therapeutic potential for liver diseases or damage by in utero transplantation.

Multiorgan engraftment and differentiation of human cord blood CD34+ Lin- cells in goats assessed by gene expression profiling.

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.

Identification and characterization of engrafted human cells in human/goat xenogeneic transplantation chimerism.

We have injected human CD34+lin- cells derived from cord blood (CB) into the goat fetuses via in utero at 45-55 days gestation under guidance of B-scan ultrasonograph. Sixty out of 68 fetuses injected survived to full term. The long-term survival of the human cells in transplant goat has been tested by various experimental methods, including FACS analysis, real-time PCR, RT-PCR, Southern-blot hybridization, FISH, as well as immunohistochemical assays. All the 60 transplant goats demonstrated engrafted human cells, including myeloid, B-lymphoid, and erythroid lineages. The yield of the human CD34+ cells varied, but was not linked with sex and age. High numbers of human cells could be detected for at least 16 months after birth. Immunohistochemical analyses revealed that the human cells were present not only in blood but also in other tissues, such as liver, of the transplant goats. In addition, a human-specific serum albumin and the hepatocyte nuclear factor (hHNF-3beta) mRNAs specific to human hepato-antigen could be readily detected in the livers of the transplant goats. Our results demonstrate that this in utero xenograft model should be useful for expansion of human HSC and possibly for the evaluating the effectiveness of prenatal treatment of human genetic diseases.

In utero transplantation of human hematopoetic stem cells into fetal goats under B-type ultrasonographic scan: an experimental model for the study of potential prenatal therapy.

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.

[Analysis of human cells in transplanted goats using fluorescence in situ hybridization].

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.

[The expression of human specific proteins in liver tissue of chimeric goats engrafted with human hematopoitic stem cells].

OBJECTIVE: To investigate the expression of human specific proteins in liver tissue obtained from goats engrafted with human hematopoietic stem cells (hHSC). METHODS: hHSCs derived from cord blood were transplanted into fetal goats for production of chimerism. Liver specimens were obtained from four 10-month-old chimeric goats and examined for their reactivity with monoclonal antibodies against human antigens: proliferating cell nuclear antigen (PCNA) and hepatocyte specific antigen (HSA). The presence of human HSA positive cells in the goat liver tissue was determined by fluorescence assisted cell sorting (FACS). The expression of human hepatocyte nuclear factor (hHNF-3beta) and human serum albumin (hALB) mRNAs was determined by RT-PCR. Meanwhile, FISH experiment was also performed to detect the human cells in the chimeric livers with the probe of human p17H8. RESULTS: Successful engraftment was confirmed by the detection of human blood cell chimerism in the circulation of the transplanted goats. Human PCNA and HSA were found in liver specimens of transplanted goats but not of normals. FACS analysis showed the presence of human HSA positive cells in the liver of chimeric goats. RT-PCR results demonstrated that hHNF-3beta and hALB mRNAs were specifically expressed in liver tissues of chimeric goats. FISH experiment showed two positive hybridized signals in some liver cells of the chimeric goats, indicating that human liver-like cells were present there. CONCLUSION: Goats engrafted with hHSC are capable to produce chimeric livers. Growth and propagation of human cells in the liver tissue of such transplanted goats were possible. Particularly, the human liver-like cells in chimeric goat livers had transcriptional activity specific to human hepatocytes.

[Molecular identification of human/goat xenogeneic model].

OBJECTIVE: To identify the human hematopoietic stem cells from the human/goat xenogeneic model with molecular techniques. METHODS: DNA and total RNA were extracted from 11 transplanted goat peripheral blood cells. Human CD(34), GPA and SRY genes were amplified with PCR in these samples, and CD(34), GPA mRNA transcripts were detected using RT-PCR in 5 and 6 goat peripheral blood cells, respectively. Southern blot analysis was performed in 8 goat DNAs to detect the human specific alpha-satellite sequence. Meanwhile FISH was also performed to detect the human cells in goat blood with a probe of human Y chromosome. RESULTS: Human CD(34) and GPA genes could be detected with PCR in all the 11 goats, and SRY gene did in 5 goats transplanted with hematopoietic stem cells derived from male human babies. Southern blot showed that human specific alpha-satellite sequence was present in 8 goats. By RT-PCR, human CD(34) mRNA was detected in 5 experimental goats, GPA mRNA was found in the other 6 experimental goats and FISH assay showed that some peripheral blood cells of the human/goat xenogeneic model were positive. CONCLUSION: Existence of human cells in the recipient goats was identified by molecular detection, which was feasible for the examination of human/goat xenogeneic models.

[A study of inductive effect of hemin on expression of the beta-globin genes in K562 cells].

The recombinant plasmid HG was constructed,in which the reporter gene encoding the enhanced green fluorescent protein (EGFP) was driven by the beta-globin promoter and regulated under the HS2 element. The inductive effect of hemin on the expression of the beta-globin gene and transiently transfected beta-globin genes in K562 cells was analysed by FACS as well as RT-PCR method. The results showed that the level of gamma and beta-globin gene mRNA in K562 cells increased significantly after 24,48 and 72 hours induced with 30 micromol/LHm.And this inductive effect was stronger after 24 and 48 hours. Furthermore,the transient expression of plasmid HG in K562 cells increased significantly with hemin induction. These results indicated that the mechanism of inductive erythroid differentiation with hemin may be correlated with mechanism of gamma-->beta-globin gene.