Y-chromosome Specific Probe Research Articles

Non-invasive prenatal diagnosis of foetal gender through maternal circulation in first trimester of pregnancy

Owing to the lack of perfect accuracy and sufficient sample size in previously performed studies on cell-free foetal DNA (cffDNA) for detection of foetal gender through maternal plasma, this study aimed to investigate the efficiency of using two Y-chromosome specific probes in foetal sex determination during first trimester of pregnancy. Five millilitres of whole blood was drawn from 192 pregnant women (10–12 weeks) and was subjected to isolate cffDNA following separation of plasma. TaqMan Real-time PCR was performed on isolated cffDNA using primer pairs and probes specific for SRY, ZFY and β-globin genes. Co-amplification of ZFY and SRY genes was detected in 103 samples confirmed after the birth. Sensitivity and specificity of the test were calculated to be 100%. Further study on larger sample size is required to confirm the reproducibility of the present test in early and non-invasive determination of foetal sex.IMPACT STATEMENTWhat is already known on this subject? Foetal gender analysis through maternal plasma has been investigated in some cell-free foetal DNA (cffDNA) analysis. However, the detection rate and method of cffDNA analysis were different among various studies.What do the results of this study add? This study introduced a modified simple probe based real time analysis with perfect detection rate.What are the implications of these findings for clinical practice and/or further research? The proposed method can be used as diagnostic test in all laboratories around the world using real-time PCR to non-invasively determine the foetal gender in the initial weeks of pregnancy following confirmation in larger sample size.

Distribution pattern following systemic mesenchymal stem cell injection depends on the age of the recipient and neuronal health

BackgroundMesenchymal stem cells (MSCs) show therapeutic efficacy in many different age-related degenerative diseases, including Alzheimer’s disease. Very little is currently known about whether or not aging impacts the transplantation efficiency of MSCs.MethodsIn this study, we investigated the distribution of intravenously transplanted syngeneic MSCs derived from young and aged mice into young, aged, and transgenic APP/PS1 Alzheimer’s disease mice. MSCs from male donors were transplanted into female mice and their distribution pattern was monitored by PCR using Y-chromosome specific probes. Biodistribution of transplanted MSCs in the brains of APP/PS1 mice was additionally confirmed by immunofluorescence and confocal microscopy.ResultsFour weeks after transplantation into young mice, young MSCs were found in the lung, axillary lymph nodes, blood, kidney, bone marrow, spleen, liver, heart, and brain cortex. In contrast, young MSCs that were transplanted into aged mice were only found in the brain cortex. In both young and aged mouse recipients, transplantation of aged MSCs showed biodistribution only in the blood and spleen. Although young transplanted MSCs only showed neuronal distribution in the brain cortex in young mice, they exhibited a wide neuronal distribution pattern in the brains of APP/PS1 mice and were found in the cortex, cerebellum, hippocampus, olfactory bulb, and brainstem. The immunofluorescent signal of both transplanted MSCs and resident microglia was robust in the brains of APP/PS1 mice. Monocyte chemoattractant-1 levels were lowest in the brain cortex of young mice and were significantly increased in APP/PS1 mice. Within the hippocampus, monocyte chemoattractant-1 levels were significantly higher in aged mice compared with younger and APP/PS1 mice.ConclusionsWe demonstrate in vivo that MSC biodistribution post transplantation is detrimentally affected by aging and neuronal health. Aging of both the recipient and the donor MSCs used attenuates transplantation efficiency. Clinically, our data would suggest that aged MSCs should not be used for transplantation and that transplantation of MSCs into aged patients will be less efficacious.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

X-Y aneuploidy rate in sperm of two “minor” breeds of cattle (Bos taurus) by using dual color fluorescent in situ hybridization (FISH)

The present study reports on the frequency of X-Y aneuploidy in the sperm population of two minor cattle breeds reared in Italy, namely Modicana and Agerolese, which are listed in the “Anagraphic Register of autochthonous cattle populations with limited distribution”. More than 50 000 sperm nuclei from 11 subjects (5 and 6, respectively for each breed) have been analyzed by the fluorescent in situ hybridization with the Xcen and Y-chromosome specific painting probes. The fraction of X- and Y-bearing sperm was close to the 1:1 ratio in the Modicana breed, whereas in the Agerolese the Y-fraction was significantly higher (P < 0.002) compared to the X-counterpart. The mean rates of X-Y aneuploidy were 0.510 and 0.466%, respectively, in the two breeds; no significant differences were found among individual bulls within each breed. Average frequencies of disomic and diploid sperm were 0.425 and 0.085% in the former and 0.380 and 0.086% in the latter. In both breeds, (a) disomy was significantly more frequent than diploidy (P < 0.01), (b) YY disomy was significantly (P < 0.001) more frequent than XY or XX; (c) MI errors (XY disomy) were significantly (P < 0.01) less represented than MII (XX + YY disomy). Compared to the dairy (Italian Friesian and Brown) and meat (Podolian and Maremmana) breeds previously analyzed, the “minor” breeds investigated in the present study showed a significantly (P < 0.002) higher rate of X-Y aneuploidy (0.486 vs. 0.159 and 0.190%, respectively). Considering all the breeds analyzed -so far- and assuming no significant interchromosomal effect, the baseline level of aneuploidy in the sperm population of the species Bos taurus was estimated as 5.19%. Establishing the baseline level of aneuploidy in the sperm population of the various livestock species/breeds engaged in animal production could reveal useful for monitoring future trends of their reproductive health, especially in relation to management errors and/or environmental hazards.

80 RECIPIENT SERUM PROGESTERONE CONCENTRATION AND EARLY CONCEPTUS DEVELOPMENT IN BOVINE EMBRYOS PRODUCED IN VIVO OR IN VITRO

The majority of pregnancy loss in cattle occurs during the first 2 to 3 weeks of pregnancy. This loss can be studied by the transfer of in vivo- and in vitro-produced embryos. The objective of this study was to examine the relationship between recipient serum progesterone levels both at the time of embryo transfer and at conceptus recovery on conceptus development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing (IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single grade-1 blastocysts from each embryo production system were transferred into heifers at Day 7. Conceptuses were recovered at Day 17 of gestation (number recovered/number transferred: IVO, 11/17; IVPS, 16/27; IVPSR, 18/34) and classified as complete, degenerated, or no conceptus. Recipient serum progesterone concentrations were determined by radioimmunoassay and compared with conceptus development outcomes. Sex of conceptus was determined by PCR using a Y-chromosome specific probe. Data were analysed using Fisher's exact test or ANOVA and Duncan's multiple range test. Compared with the IVO group, in vitro-produced embryos had more (P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; IVPSR, 20.6%). There were no differences (P &gt; 0.05) in serum progesterone concentrations in recipients assigned to different treatments at Day 7. There was also no effect (P &gt; 0.05) of treatment on progesterone levels in recipients with either male or female conceptuses at the time of transfer. Interestingly, heifers in the in vitro treatment groups had lower (P &lt; 0.01) progesterone concentrations at Day 7 when no conceptus was recovered at Day 17 (IVPS, 2.1 ± 0.4 ng mL–1; IVPSR, 2.7 ± 0.4 ng mL–1; Least squares means ± standard error of the mean) compared with the IVO group (4.5 ± 0.6 ng mL–1). There was no difference in progesterone concentration between treatment groups for heifers with shorter conceptuses (≤194 mm). However, when longer (&gt;194 mm) conceptuses were recovered, heifers with in vitro produced embryos had lower (P &lt; 0.05) progesterone levels at Day 7 compared with those with in vivo produced embryos (IVPS, 2.2 ± 0.6 ng mL–1; IVPSR, 2.3 ± 0.5 ng mL–1; IVO, 3.9 ± 0.6 ng mL–1). In summary, serum progesterone concentrations in recipients at the time of transfer of in vivo- or in vitro-produced embryos were associated with conceptus development at Day 17 of gestation. Research supported by NC State University GAANN Biotechnology Fellowship (C. V. Barnwell) and the College of Veterinary Medicine.

BOVINE EMBRYO SEXING USING THE FLUORESCENCE IN SITU HYBRIDIZATION (FISH)

The purpose of this study was to elaborate a Y-chromosome specific DNA probe that can be used in order to efficiently sex the bovine embryos using biopsied blastomeres. Another objective was to evaluate the pregnancy rate in recipient cows in which the biopsied and sexed embryos were transferred. A number of 76 bovine embryos have been non-surgically collected from a batch of 8 Simmental donor cows, following superovulation and artificial insemination. The embryos have been morphologically evaluated and biopsy has been performed in order to collect a small number of blastomeres from the inner cell mass. After the biopsy, the embryos have been immediately transferred to recipient cows, whose estrous cycle had previously been synchronized with the donor cows. The biopsied blastomeres were treated with 1 µg/ml vinblastine sulphate for 6 hours in order to induce the chromosomal metaphases, fixed on a slide using methanol and acetic acid and then kept in the freezer until use. The DNA probe has been synthesized as follows: the sequence of the Y-chromosome specific BtY2 gene has been used in order to obtain male specific primers; the primers were used in order to amplify a male specific DNA sequence obtained from a bull’s blood; after amplification and electrophoresis the specific bands were cut and DNA has been extracted from the gel; the male specific probe has subsequently been biotinylated by nick translation. Using this probe FISH has been performed and fluorescent signal has been amplified using the tyramide signal amplification kit. Slides have been examined using a fluorescence microscope (the presence of fluorescence signifying a male embryo while its absence a female embryo). The pregnancy rate has been evaluated by rectal palpation, 60 days after the transfer of biopsied embryos and was of 51.3%, while the accuracy of embryo sexing was evaluated at birth, when the FISH predicted sex has been compared with the morphological sex of the offspring and was of 92%. In conclusion, the fluorescence in situ hybridization method of embryo sexing turned out to be quite difficult but yielded good results and can be used for bovine embryo sexing.

Transplantation of Schwann cells and/or olfactory ensheathing glia into the contused spinal cord: Survival, migration, axon association, and functional recovery

Schwann cells (SCs) and olfactory ensheathing glia (OEG) have shown promise for spinal cord injury repair. We sought their in vivo identification following transplantation into the contused adult rat spinal cord at 1 week post-injury by: (i) DNA in situ hybridization (ISH) with a Y-chromosome specific probe to identify male transplants in female rats and (ii) lentiviral vector-mediated expression of EGFP. Survival, migration, and axon-glia association were quantified from 3 days to 9 weeks post-transplantation. At 3 weeks after transplantation into the lesion, a 60-90% loss of grafted cells was observed. OEG-only grafts survived very poorly within the lesion (<5%); injection outside the lesion led to a 60% survival rate, implying that the injury milieu was hostile to transplanted cells and or prevented their proliferation. At later times post-grafting, p75(+)/EGFP(-) cells in the lesion outnumbered EGFP(+) cells in all paradigms, evidence of significant host SC infiltration. SCs and OEG injected into the injury failed to migrate from the lesion. Injection of OEG outside of the injury resulted in their migration into the SC-injected injury site, not via normal-appearing host tissue but along the pia or via the central canal. In all paradigms, host axons were seen in association with or ensheathed by transplanted glia. Numerous myelinated axons were found within regions of grafted SCs but not OEG. The current study details the temporal survival, migration, axon association of SCs and OEG, and functional recovery after grafting into the contused spinal cord, research previously complicated due to a lack of quality, long-term markers for cell tracking in vivo.

Influence of Stem Cell Mobilization and Liver Regeneration on Hepatic Parenchymal Chimerism in the Rat

Despite the newly discovered plasticity of hematopoietic stem cells, their capacity to "transdifferentiate" into parenchymal cells and the regulation of this process is not yet elucidated. The present study was designed to investigate the effect of stem cell mobilization and liver regeneration on this process using a syngeneic rat female-to-male liver transplantation model. Rate and frequency of Y-chromosome containing hepatocytes was determined by fluorescence-in situ hybridization (FISH) using a rat Y-chromosome specific probe. Rats were subjected to full-size or partial liver transplantation with and without stem cell mobilization using granulocyte colony stimulating factor (G-CSF). The effect of stem cell mobilization was confirmed by assessing the white blood count and by evaluating the migration of granulopoietic precursor cells to the liver. Treatment with G-CSF induced a twofold increase of peripheral white blood cells and a strong influx of granulopoietic precursor cells into the liver. Transplantation of a partial liver graft was followed by a 90% recovery of the original liver weight within a week, demonstrating the strong regenerative stimulus. Irrespective of the experimental model, the incidence and rate of Y-chromosome containing hepatocytes never exceeded 0.77%. Neither stem cell mobilization nor liver regeneration enhanced the incidence or rate of stem cell derived hepatocytes in a liver graft with unimpaired regenerative potential.

136. Recombinant Adeno-Associated Virus 2-Mediated Ectopic Expression of α4β1 Integrin in Mouse Mesenchymal Stem Cells Enhances Repopulation to Bone upon Syngeneic Transplantation in an Immunocompetent Mouse Model

Ex-vivo therapy with mesenchymal stem cells (MSC) is a novel approach for the treatment of genetic, age-related or segmental defects to increase bone mass. Recent identification that MSC differentiate into lineages that form bone, cartilage, adipose tissue, fibroblasts, neurons and cardiac tissues under controlled conditions offers unique advantage for potentially utilizing them in cell therapy or gene therapy. The pluripotent property of MSC necessitates selection of lineage-specific cells or restricted expression of therapeutic genes into committed progenitors with specific phenotypes for regulated gene therapy. Despite the potential of MSC to differentiate into desired lineages under controlled culture conditions, major limitation in the utilization of the culture-expanded MSC remains in the inability to restrict homing to bone upon autologous transfer. More recently, it is becoming increasingly evident that homing of hematopoietic stem cells to bone is critically dependent on the expression of matrix associated proteins, which interact with receptors on cells in the bone marrow. One of the matrix proteins that has been shown to effect significant influence on homing of not only hematopoietic stem cells but also of metastatic tumor cells to bone is the |[alpha]|4|[beta]|1integrin. |[alpha]|4|[beta]|1 integrin is a cell surface heterodimer, which mediates cell-cell and cell-extracellular matrix interactions. Towards developing conditions that will promote homing of the ex vivo modified MSC to bone and to test the hypothesis that enrichment of progenitor phenotype and homing signal on ex vivo cultured MSC, will enhance homing to bone upon systemic transplantation, bone marrow stromal cells from male C57BL/6 mouse were transduced with a recombinant AAV2 encoding mouse |[alpha]|4|[beta]|1 integrin. The cells were cultured for 10 days in the presence of 10-9 M FGF2 to avoid differentiation. Approximately 107 cells were transplanted into female recipients by tail vein injection. Cohorts of mice also received untransduced or rAAV2-GFP transduced MSC. Animals were sacrificed 15days post transplantation and tissues including lung, liver, adipose tissues, spleen, kidney, muscle, lymph node and bone marrow were harvested. FACS analysis of cultured bone marrow stromal cells from control and experimental mice, in situ hybridization of tissue with Y-chromosome specific probe, real-time PCR and southern blot analyses established significantly higher percentage of bone homing of culture-expanded MSC, transduced with rAAV expressing |[alpha]|4|[beta]|1 integrin. These studies indicate that rAAV encoding a4b1integrin can be effectively used to transduce MSC ex vivo to achieve enhanced repopulation of gene-modified MSC to bone, and can be combined with other cell/gene therapy applications for targeted delivery of therapeutic proteins.

Effects of plastic-adherent dermal multipotent cells on peripheral blood leukocytes and CFU-GM in rats

Multipotent cells were isolated from the dermis of newborn rats by their adherence to plastic culture dishes. Morphological observations showed that most of these elements were fibroblast-like cells described as cultured dermal multipotent cells (DMCs). These cells express CD90, CD44, CD59, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). They display the capacity to differentiate into cells with the phenotypic properties of osteocytes, adipocytes, chondrocytes, or neurons depending upon the specific inducing media. The confluent dermal multipotent cell layer supported and promoted the in vitro growth of colony-forming unit of granulocyte/macrophage progenitors (CFU-GM) as well as hematopoietic progenitor colonies. Transplantation of dermal multipotent cells into sublethally irradiated rats led to a significant increase of peripheral blood white cells nucleated cells, and CFU-GM colonies in the bone marrow. Fluorescence in situ hybridization analysis using a Y-chromosome–specific probe showed that dermal multipotent cells could engraft into bone marrow. However, they failed to differentiate into CD45-positive hematopoietic cells and to repopulate the hematopoietic system of lethally irradiated rats. These results suggest that plastic-adherent DMCs may at least represent an alternative source of mesenchymal stem cells to restore the marrow microenvironment and promote the survival, engraftment, and proliferation of hematopoietic cells.

Transplantation of dermal multipotent cells promotes the hematopoietic recovery in sublethally irradiated rats.

Our previous study indicated that dermal multipotent cells with the differentiation capacity to form cells with the phenotypic properties of osteocytes, adipocytes, chondrocytes, and neurons in specific inducing media could be isolated from the enzymatically dissociated dermal cells of newborn rats by their adherence to culture dish plastic. We have also observed that the systemic transplantation of dermal multipotent cells could not repopulate the hematopoietic system in lethally irradiated rats. In this paper, we found that a transplantation of plastic-adherent dermal multipotent cells into sublethally irradiated rats led to a significant increase of white blood cells in peripheral blood, nucleated cells, CFU-GM, and CFU-F colonies in bone marrow. FISH analysis, using a Y-chromosome specific probe, showed that dermal multipotent cells could engraft into bone marrow in recipients. Flow cytometry (FACS) analysis also showed that the proportion of CD2 and CD25 positive lymphocytes in peripheral blood did not change significantly in two weeks after transplantation. By these results, we infer that dermal multipotent cells may represent an alternative origin of mesenchymal stem cells to restore marrow microenvironment and promote the survival, engraftment, and proliferation of hematopoietic cells.

A role for natural killer cells in the rapid death of cultured donor myoblasts after transplantation.

The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts. Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice. A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells. These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.

Quantitative polymerase chain reaction-based assay with fluorogenic Y-chromosome specific probes to measure bone marrow chimerism in mice

A quantitative duplex polymerase chain reaction (PCR) method using the LightCycler™ detection system was developed to generate a reproducible and convenient method to quantitatively measure chimeric states in murine transplantational models using male and female BALB/c mice. Materials and methods: DNA mixtures isolated from male and female murine BALB/c bone marrow cells were analyzed with Y-chromosome and control autosomal GAPDH specific primers by either monoplex or duplex real-time quantitative PCR using the LightCycler™ detection system to determine chimeric states. Results: High specific Y-chromosome and control autosomal GAPDH primers gave a detection sensitivity of approximately 0.01% using LightCycler™ PCR. Analysis of standard curve distribution of diluted DNA samples accurately matched the proportional dilutions. Y6 primers run on male DNA samples against a female background showed amplification slope initiations reflecting the amounts of male DNA contained in these mixtures. Mixed samples run with GAPDH primers showed amplification corresponding to 100% DNA amounts of limiting dilution runs which were used as controls. Calculated engraftment generated by monoplex PCR lacked reproducibility. Quantitation using duplex PCR using specific detection probes for both Y-chromosome Y6 and autosomal GAPDH primer amplicons allowed precise and reproducible calculations of engraftment levels. Conclusion: Duplex PCR using specific detection probes for Y-chromosome Y6 and autosomal GAPDH primer amplicons allows for rapid, precise and specific engraftment determination after transplantation of male BALB/c marrow cells into female BALB/c recipients.

Complement and myoblast transfer therapy: donor myoblast survival is enhanced following depletion of host complement C3 using cobra venom factor, but not in the absence of C5.

Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne Muscular Dystrophy and involves the injection of cultured muscle precursor cells ('myoblasts') isolated from normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT is widely accepted as being due to the immune response of the host. The role of complement as one possible mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement. Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by 3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3 increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approximately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast survival by a mechanism possibly involving activated C3 fragments.

Prenatal screening of single-gene disorders from maternal blood.

Fetal cells and cell-free fetal DNA can be found circulating in maternal blood. Fetal cells recovered from maternal blood provide the only source of pure fetal DNA for noninvasive prenatal DNA diagnosis. Fetal nucleated erythrocytes (NRBCs) are considered the most suitable maternally-circulating fetal cells for this purpose, because they are not commonly found in the peripheral blood of healthy adults and are most abundant in the fetus during early gestation. Because fetal cells in maternal blood are extremely rare, a definitive separation method has not yet been established. Fetal NRBCs can be enriched from maternal blood via fluorescence- or magnetic-activated cell sorting, density gradients, immuno-magnetic beads or micromanipulation. Fetal cells are identified by Giemsa staining, hybridization with Y-chromosome specific probes, PCR-detection of a specific paternal allele, or immunostaining for fetal cell antigens. Amplification of fetal DNA sequences by primer extension preamplification and PCR has allowed prenatal screening for Duchenne muscular dystrophy and the fetal RhD blood type. Sequence-specific hybridization has been used to detect sickle cell anemia and beta-thalassemia prenatally in heterozygous carriers of these disorders. The use of cell-free fetal DNA in maternal plasma for the diagnosis of single-gene disorders is limited to disorders caused by a paternally inherited gene or a mutation that can be distinguished from the maternally inherited counterpart. At present, fetal gender can be determined from maternal plasma. When a pregnant woman is a heterzygous carrier of an X-linked disorder, the determination of fetal gender is clinically very informative for first-step screening to avoid invasive amniocentesis. The non-invasive prenatal diagnosis of genetic disorders should be applied to pregnant women with a definite risk for a specific single-gene disorder.

The use of transferrin for enrichment of fetal cells from maternal blood

Iron loaded transferrin (holotransferrin) was used for enrichment of fetal cells from peripheral blood of pregnant women. Cord blood samples were used to evaluate enrichment efficacy of single and double MACS separations. Blood samples were obtained from 10 pregnant women prior to chorion villus sampling (CVS). Erythroblasts and other mononuclear cells were isolated by triple-density gradient centrifugation. Fetal cells were further enriched by positive magnetic sorting (VarioMACS) using biotinylated transferrin and streptavidin conjugated to magnetic microbeads. The isolated cells were analysed with dual-colour in situ hybridization (FISH) with X- and Y-chromosome specific probes. Male fetuses were correctly identified in three out of four (75%) pregnancies and female fetuses in six out of six (100%) pregnancies. By using transferrin instead of antibodies to the transferrin receptor to label and enrich fetal cells, we believe that unspecific binding attributed to immunological labelling can be minimized. The results indicate that transferrin may be an alternative to antibodies to transferrin receptor for separation of fetal cells from maternal blood.

Enhanced migration and fusion of donor myoblasts in dystrophic and normal host muscle

Sliced male C57Bl/10Sn (H2-b) donor muscles were grafted into the female histocompatible muscles of untreated, FK506-treated, and T-cell depleted (with or without thymic tolerization) dystrophic (mdx; H2-b) and normal (C57Bl/10Sn; H2-b) hosts, and also into histoincompatible normal (Balb/c; H2-d) hosts. The fate of male donor nuclei was monitored on tissue sections by in situ hybridization with a Y-chromosome specific probe. The results demonstrate that the dystrophic environment is more conducive than normal muscle to donor myoblast migration, with the distance moved being threefold greater at 12 weeks in dystrophic hosts. T-cell depletion was significantly more effective than FK506 treatment at enhancing donor myoblast emigration in both histocompatible and histoincompatible hosts at 3 weeks. Furthermore, the effects of T-cell depletion were sustained in histoincompatible hosts at 12 weeks. These data endorse the use of host T-cell depletion as a promising long-term strategy to improve myoblast transfer therapy (MTT) in the clinical situation. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 560–574, 2000.

Enhanced migration and fusion of donor myoblasts in dystrophic and normal host muscle.

Sliced male C57Bl/10Sn (H2-b) donor muscles were grafted into the female histocompatible muscles of untreated, FK506-treated, and T-cell depleted (with or without thymic tolerization) dystrophic (mdx; H2-b) and normal (C57Bl/10Sn; H2-b) hosts, and also into histoincompatible normal (Balb/c; H2-d) hosts. The fate of male donor nuclei was monitored on tissue sections by in situ hybridization with a Y-chromosome specific probe. The results demonstrate that the dystrophic environment is more conducive than normal muscle to donor myoblast migration, with the distance moved being threefold greater at 12 weeks in dystrophic hosts. T-cell depletion was significantly more effective than FK506 treatment at enhancing donor myoblast emigration in both histocompatible and histoincompatible hosts at 3 weeks. Furthermore, the effects of T-cell depletion were sustained in histoincompatible hosts at 12 weeks. These data endorse the use of host T-cell depletion as a promising long-term strategy to improve myoblast transfer therapy (MTT) in the clinical situation.

DNA Content and Ploidy Level of Bovine Placentomal Trophoblast Giant Cells

Cytophotometric measurement of the DNA content of Feulgen-stained nuclei in touch preparations of bovine placentomes (n=5) revealed that 8C nuclei occurred in all, 16C nuclei in two, and 32C nuclei in one specimen. The determination of ploidy level by in situ hybridization with a Y-chromosome specific DNA probe showed that the majority of the fetal nuclei in touch preparations of placentomes from male fetuses (n=5) are tetraploid. Generally two tetraploid nuclei lie close together. These findings indicate that polyploidization is a normal feature in the development of the mostly binucleate trophoblast giant cells (TGCs). A new model for the development of these cells is proposed: a primary acytokinetic mitosis leads to a binucleate cell with two diploid nuclei. This cell enters a second acytokinetic mitosis during which the chromosomes of both nuclei form a common metaphase plate. The resulting cell with two tetraploid nuclei undergoes an additional S-phase but does not enter a renewed mitosis.The functional significance of this genome multiplication may be an increased synthetic capacity of bovine TGCs, caused by an increased number of gene copies available for transcription. Since genome multiplication is a property of invasive trophoblast cells of different species, it may be advantageous for trophoblast invasion.

In Vivo Identification, Survival, and Functional Efficacy of Transplanted Hepatocytes in Acute Liver Failure Mice Model by FISH Using Y-Chromosome Probe

Hepatocyte transplantation has excited much interest in lending temporary metabolic support to a failing liver following acute liver injury. The exact site from which they act and the clinical, biochemical, and histological changes in the recipient body following hepatocyte transplantation is yet to be worked out. The present study is an attempt to delineate location and function of transplanted hepatocytes and also the overall survival of these cells with a fluorescent in situ hybridization (FISH) technique using a Y-chromosome–specific probe in a carbon tetrachloride (CCl 4)-induced mice model of fulminant hepatic failure. Fifty-five syngenic adult Swiss female mice of approximately the same age and body weight were divided into three groups. Group-1 ( n = 15), which received mineral oil, served as a negative control. Group-II ( n = 15) received CCl 4 (3 mL/kg) 40% vol/vol in mineral oil, by gavage served as positive control for hepatic failure. Group-III ( n = 25) received intrasplenic transplantation of syngenic single cell suspension of hepatocytes in Hanks medium, after 30 h of CCl 4 administration. Male Swiss adult mice ( n = 15) served as donors of hepatocytes. The overall survival of animals in groups I to III was 100, 0, and 70%, respectively, by 2 wk of the study period. Transplanted hepatocytes were identified by Periodic Acid Schiff (PAS) staining and confirmed with a FISH technique using the Y-chromosome probe. The majority of exogenously transplanted hepatocytes were found in the liver and spleen sections even after 1 wk of hepatocyte transplantation. Transplanted cells were mostly found to be translocated into the sinusoids of the liver. Transplanted hepatocytes were found to be beneficial as a temporary liver support in a failing liver, significantly improving the survival of the animals. In the present study, the FISH technique was used to unequivocally distinguish the transplanted cells from the host, and thus describes a model for studying the distribution and survival of the transplanted cells.