Jingly F. Weier

The human placenta is a hematopoietic organ during the embryonic and fetal periods of development

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations-CD34(++)CD45(low) and CD34(+)CD45(low)-that were found in chorionic villi and in the chorioamniotic membrane. CD34(++)CD45(low) cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34(++)CD45(low) cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56(+) natural killer cells and CD19(+)CD20(+)sIgM(+) B cells in polyclonal liquid cultures. CD34(+)CD45(low) cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34(-) cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34(++)CD45(low) cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5-8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

Poor development of human nuclear transfer embryos using failed fertilized oocytes

Failed fertilized human oocytes from IVF were enucleated and used as recipients for somatic cell nuclear transfer. The reconstructed embryos frequently formed an expanded nucleus from the injected genome after activation. However, subsequent development beyond the 1-cell stage was poor, and the resulting embryos showed chromosomal abnormalities. Poor development of oocytes after nuclear transfer contrasted with that of control, sperm-injected oocytes, which often progressed to cleavage stages. These results suggest that failed fertilized oocytes are not effective recipients for somatic cell nuclear transfer.

DNA Probe Pooling for Rapid Delineation of Chromosomal Breakpoints

Structural chromosome aberrations are hallmarks of many human genetic diseases. The precise mapping of translocation breakpoints in tumors is important for identification of genes with altered levels of expression, prediction of tumor progression, therapy response, or length of disease-free survival, as well as the preparation of probes for detection of tumor cells in peripheral blood. Similarly, in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD) for carriers of balanced, reciprocal translocations benefit from accurate breakpoint maps in the preparation of patient-specific DNA probes followed by a selection of normal or balanced oocytes or embryos. We expedited the process of breakpoint mapping and preparation of case-specific probes by utilizing physically mapped bacterial artificial chromosome clones. Historically, breakpoint mapping is based on the definition of the smallest interval between proximal and distal probes. Thus, many of the DNA probes prepared for multiclone and multicolor mapping experiments do not generate additional information. Our pooling protocol, described here with examples from thyroid cancer research and PGD, accelerates the delineation of translocation breakpoints without sacrificing resolution. The turnaround time from clone selection to mapping results using tumor or IVF patient samples can be as short as 3 to 4 days.

Chromosome-specific DNA Repeat Probes

In research as well as in clinical applications, fluorescence in situ hybridization (FISH) has gained increasing popularity as a highly sensitive technique to study cytogenetic changes. Today, hundreds of commercially available DNA probes serve the basic needs of the biomedical research community. Widespread applications, however, are often limited by the lack of appropriately labeled, specific nucleic acid probes. We describe two approaches for an expeditious preparation of chromosome-specific DNAs and the subsequent probe labeling with reporter molecules of choice. The described techniques allow the preparation of highly specific DNA repeat probes suitable for enumeration of chromosomes in interphase cell nuclei or tissue sections. In addition, there is no need for chromosome enrichment by flow cytometry and sorting or molecular cloning. Our PCR-based method uses either bacterial artificial chromosomes or human genomic DNA as templates with α-satellite-specific primers. Here we demonstrate the production of fluorochrome-labeled DNA repeat probes specific for human chromosomes 17 and 18 in just a few days without the need for highly specialized equipment and without the limitation to only a few fluorochrome labels.

Primary cutaneous perivascular epithelioid cell tumor: A clinicopathological and molecular reappraisal

BACKGROUND Perivascular epithelioid cell tumor (PEComa) is a rare neoplasm of uncertain histogenesis with a mixed myomelanocytic immunophenotype, rarely arising in the skin (primary cutaneous PEComa [pcPEComa]). OBJECTIVE We analyzed the clinicopathological features of 8 pcPEComas, assayed for DNA copy number changes and for initiating mutations common in melanocytic neoplasms. METHODS pcPEComas were evaluated using immunohistochemistry, comparative genomic hybridization, and DNA sequencing. RESULTS pcPEComas were erythematous nodules, mostly in the lower extremities of women (5/8), composed of large pale-staining epithelioid cells. The patients age range was 26 to 67 (mean 46) years. The percentages of tumors staining positively were as follows: micro-ophthalmia-associated transcription factor, NKI/C3, bcl-1, E-cadherin, and cathepsin K (100%); HMB-45, 4E-binding protein 1, and CD68 (88%); smooth muscle actin and muscle-specific actin (40%); S100 (38%); calponin (20%); desmin (13%); and melan-A, SOX10, and keratin (0%). No chromosomal copy number changes or initiating mutations were identified. LIMITATIONS Small sample size is a limitation. CONCLUSIONS pcPEComas have a different molecular signature than extracutaneous tumors and are unrelated to tuberous sclerosis. However, the common expression of 4E-binding protein 1 points to a role of the mTOR pathway in their pathogenesis. Because pcPEComas are diagnostically challenging, we propose that micro-ophthalmia-associated transcription factor, NKIC3, smooth muscle actin, desmin, bcl-1, cathepsin K, and 4E-binding protein 1 can be used when evaluating a possible pcPEComa.

Fluorescence in situ hybridization and spectral imaging analysis of human oocytes and first polar bodies.

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35–39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis.

Molecular cytogenetic studies towards the full karyotype analysis of human blastocysts and cytotrophoblasts.

Numerical chromosome aberrations in gametes typically lead to failed fertilization, spontaneous abortion or a chromosomally abnormal fetus. By means of preimplantation genetic diagnosis (PGD), we now can screen human embryos in vitro for aneuploidy before transferring the embryos to the uterus. PGD allows us to select unaffected embryos for transfer and increases the implantation rate in in vitro fertilization programs. Molecular cytogenetic analyses using multi-color fluorescence in situ hybridization (FISH) of blastomeres have become the major tool for preimplantation genetic screening of aneuploidy. However, current FISH technology can test for only a small number of chromosome abnormalities and hitherto failed to increase the pregnancy rates as expected. We are in the process of developing multi-color FISH-based technologies to score all 24 chromosomes in single cells within a three-day time limit, which we believe is vital to the clinical setting. Also, human placental cytotrophoblasts (CTBs) at the fetal-maternal interface acquire aneuploidies as they differentiate to an invasive phenotype. About 20–50% of invasive CTB cells from uncomplicated pregnancies were found to be aneuploid, suggesting that the acquisition of aneuploidy is an important component of normal placentation, perhaps limiting the proliferative and invasive potential of CTBs. Since most invasive CTBs are interphase cells and possess extreme heterogeneity, we applied multi-color FISH and repeated hybridizations to investigate the feasibility of a full karyotype analysis of individual CTBs. In summary, this study demonstrates the strength of Spectral Imaging analysis and repeated hybridizations, which provides a basis for full karyotype analysis of single interphase cells.

Preimplantation genetic diagnosis for gender selection in the USA

Preimplantation genetic diagnosis (PGD) for gender selection for non-medical reasons has been considered an unethical procedure by several authors and agencies in the Western society on the basis that it could disrupt the sex ratio, that it discriminates against women and that it leads to disposal of normal embryos of the non-desired gender. In this study, the analysis of a large series of PGD procedures for gender selection from a wide geographical area in the USA shows that, in general, there is no deviation in preference towards any specific gender except for a preference of males in some ethnic populations of Chinese, Indian and Middle Eastern origin that represent a small percentage of the US population. In cases where only normal embryos of the non-desired gender are available, 45.5% of the couples elect to cancel the transfer, while 54.5% of them are open to have embryos transferred of the non-desired gender, this fact being strongly linked to cultural and ethnic background of the parents. In addition this study adds some evidence to the proposition that, in couples with previous children of a given gender, there is no biological predisposition towards producing embryos of that same gender. Based on these facts, it seems that objections to gender selection formulated by ethics committees and scientific societies are not well founded.

BAC-FISH assays delineate complex chromosomal rearrangements in a case of post-Chernobyl childhood thyroid cancer

Structural chromosome aberrations are known hallmarks of many solid tumors. In the papillary form of thyroid cancer (PTC), for example, activation of the receptor tyrosine kinase (RTK) genes, RET and neurotrophic tyrosine kinase receptor type I (NTRK1) by intra- and interchromosomal rearrangements has been suggested as a cause of the disease. However, many phenotypically similar tumors do not carry an activated RET or NTRK-1 gene or express abnormal ret or NTRK-1 transcripts. Thus, we hypothesize that other cellular RTK-type genes are aberrantly expressed in these tumors. Using fluorescence in situ hybridization-based methods, we are studying karyotype changes in a relatively rare subgroup of PTCs, i.e., tumors that arose in children following the 1986 nuclear accident in Chernobyl, Ukraine. Here, we report our technical developments and progress in deciphering complex chromosome aberrations in case S48TK, an aggressively growing PTC cell line, which shows an unusual high number of unbalanced translocations.

DNA copy number changes in tumors within the spectrum of cellular, atypical, and metastasizing fibrous histiocytoma

BACKGROUND Cutaneous fibrous histiocytoma (FH) is a common mesenchymal neoplasm. Metastasis is rare, disproportionately occurring among the aneurysmal, cellular, atypical, and deep variants. OBJECTIVE We determined whether DNA copy number changes occurred in atypical FH (AFH), and whether they were similar to those in metastasizing FH (MetFH) and benign cellular FH (CFH). METHODS Five primary tumors of MetFH were evaluated by array-based comparative genomic hybridization analysis, with tissue from local recurrences and lung metastases in 2 and 2 patients, respectively. Seven indolent AFH and 5 CFH were identified for comparison. RESULTS Substantial differences between the groups were found both in the frequency of chromosomal aberrations (higher among MetFH and absent or solitary in CFH) and array-based comparative genomic hybridization profiles (frequent gains of 7 and 8q and losses of Xq in MetFH; recurrent losses of chromosomes 9 and 22 in AFH; isolated loss of 5q and gain in chromosome 20 in 2 CFH). Fatal MetFH cases (2 of 5 cases) exhibited the highest rate of chromosomal aberrations. LIMITATIONS This study included a small sample size with a short-term follow-up. CONCLUSIONS Benign CFH, indolent AFH, and MetFH represent distinct biological entities within the spectrum of FH; array-based comparative genomic hybridization may be a tool in recognizing FH cases with metastatic potential and increasingly aggressive behavior.