Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood.

BACKGROUND: Many methods have been employed to obtain fetal cells from maternal blood for prenatal diagnostics, but there has been little work done that compares the efficacy of different methods. This study presents a comparison of two commonly used methods for selecting erythroblasts with selection directly from whole blood. METHODS: Erythroblasts were isolated from maternal blood by either differential lysis or density separation, followed by selection with an antibody to the transferrin receptor. These methods were compared with antibody selection directly from whole blood. The total yield of erythroblasts was determined for each method. RESULTS: Red cell lysis is not recommended because the lysis step cannot be well controlled. Density separation followed by antibody selection works well. However, a faster and simpler method, antibody selection directly from whole blood using Immunicon Ferrofluid and magnetic separators, works as well and has the potential to yield even more cells. CONCLUSIONS: Considering the need for a simple and quick method for selecting fetal cells from maternal blood, we suggest selection directly from whole blood.

A novel method for depositing erythroid cells onto glass slides for fetal cell analysis.

BACKGROUND: We have developed a method for selecting erythroblasts from blood, the first step toward identifying fetal cells in maternal blood for diagnostic purposes. Because the selection method results in a large number of positive cells, we needed to develop new methods to deposit the cells onto slides and to modify in situ hybridization procedures to enable detection of fetal cells. METHODS: We utilized Nunc flaskettes to increase the slide surface area available for cell deposition. The ability of erythroid lineage cells to adhere to several surface modifications was examined. In situ hybridization methods were tested to find the best approach that is compatible with these cell preparations. RESULTS: The best glass slide coating for erythroid cells was found to be an antibody to glycophorin A, a red cell surface antigen. We were able to get excellent in situ hybridization signals in cells on flaskettes by modifying fixation and pretreatment parameters. CONCLUSIONS: The methods described here appear to be the best way of attaching a large number of erythroid lineage cells to slides and of detecting them by in situ hybridization.

Inconsistency of fetal trophoblast cells in first trimester maternal peripheral blood prevents non-invasive fetal testing using this cell target.

We have investigated whether maternal peripheral blood from the first trimester of pregnancy is a reliable source of identifiable trophoblast cells. The cells were enriched from 30 ml of venous blood, with multiple antibodies shown previously to enrich trophoblasts and a new cocktail based on known trophoblast surface features. Three different magnetic solid phases were tested to enrich trophoblasts, and both positive and negative cell enrichment strategies were examined. The cells were identified as trophoblast by morphology coupled with immunocytochemistry to co-localize cytokeratin with one of three IGF-II, PAI-1 or hPLH proteins or by in-situ hybridization with a mixture of 50 oligos directed to eight different expressed genes, alpha-HCG, IGF-II, PAI-1, HASH2, hPLH, p57(KIP2), PP5, H-19. While these tools worked beautifully in chorionic villi cell/sprout preparations and tissue sections, we could not detect and identify any trophoblasts in maternal peripheral blood even if the maternal peripheral blood was drawn 5-20 min following termination of pregnancy or from individuals maintaining the pregnancy. Based on our own experience and that of some reports in the literature, trophoblasts do not appear to be a viable candidate for fetal screening using maternal peripheral blood as the source. It is important to note that while trophoblast deportation is a biological phenomenon that has been described repeatable, they do not provide a means to perform prenatal genetic diagnosis.

Enrichment and identification of fetal trophoblast cells from first trimester maternal cervical lavage and uterine blood specimens.

First trimester prenatal diagnosis of fetal aneuploidies is an active area of research despite years of disappointing data employing maternal peripheral blood samples. To remedy this situation we have investigated other first trimester maternal specimens attempting to find a consistent fetal cell source. Using our previously established positive enrichment procedure along with a commercially available depletion method, fetal trophoblast cells were identified employing immunocytochemistry using an antibody cocktail or by using mRNA in-situ hybridization employing a cocktail of trophoblast specific probes. Fetal origin of positively identified cells was verified using interphase fluorescent in-situ hybridization (FISH) for X and Y-chromosomes. Artificial model systems were established that indicated yields of trophoblast cells and allowed the enrichment procedure to be optimized for minimal losses from maternal specimens. We demonstrate herein that blood drawn from maternal vessels near the placental implantation site to be the most consistent source of fetal cells from any first trimester maternal specimen described to date. In addition, a high yield of multinucleated syncytiotrophoblast cells was obtained using a cell depletion strategy to enrich the target cells. The safety of the procedure or even the clinical utility of blood drawn from maternal vessels near the placental implantation site is yet to be demonstrated.

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.

Evidence that the homeodomain protein Gtx is involved in the regulation of oligodendrocyte myelination.

We have investigated the patterns of postnatal brain expression and DNA binding of Gtx, a homeodomain transcription factor. Gtx mRNA accumulates in parallel with the RNAs encoding the major structural proteins of myelin, myelin basic protein (MBP), and proteolipid protein (PLP) during postnatal brain development; Gtx mRNA decreases in parallel with MBP and PLP mRNAs in the brains of myelin-deficient rats, which have a point mutation in the PLP gene. Gtx mRNA is expressed in differentiated, postmitotic oligodendrocytes but is not found in oligodendrocyte precursors or astrocytes. These data thus demonstrate that Gtx is expressed uniquely in differentiated oligodendrocytes in postnatal rodent brain and that its expression is regulated in parallel with the major myelin protein mRNAs, encoding MBP and PLP, under a variety of physiologically relevant circumstances. Using a Gtx fusion protein produced in bacteria, we have confirmed that Gtx is a sequence-specific DNA-binding protein, which binds DNA sequences containing a core AT-rich homeodomain binding site. Immunoprecipitation of labeled DNA fragments encoding either the MBP or PLP promoter regions with this fusion protein has identified several Gtx-binding fragments, and we have confirmed these data using an electrophoretic mobility shift assay. In this way we have identified four Gtx binding sites within the first 750 bp of the MBP promoter and four Gtx binding sites within the first 1. 3 kb of the PLP promoter. In addition, inspection of the PLP promoter sequence demonstrates the presence of six additional Gtx binding sites. These data, taken together, strongly suggest that Gtx is important for the function of differentiated oligodendrocytes and may be involved in the regulation of myelin-specific gene expression.

Evidence for a regulatory element controlling amino acid transport system L in Chinese hamster ovary cells.

We have used the technique of somatic cell hybridization to study the regulation of the neutral amino acid transport system L in Chinese hamster ovary (CHO) cells. The cell line CHO-ts025C1 has a temperature-sensitive mutation in leucyl-tRNA synthetase. At the nonpermissive temperature of 39 degrees C, CHO-ts025C1 cells are unable to charge leucyl-tRNA and behave as though starved for leucine by increasing their system L transport activity two- to fourfold. From the temperature-sensitive cell line, we have isolated a regulatory mutant cell line, CHO-C11B6, that has constitutively elevated system L transport activity. The CHO-C11B6 cell line retains the temperature-sensitive leucyl-tRNA synthetase mutation, but growth of this cell line is temperature resistant because its increased system L transport activity leads to increased intracellular leucine levels, which compensate for the defective synthetase. Hybrid cells formed by fusion of the temperature-sensitive CHO-ts025C1 cells and the temperature-resistant CHO-C11B6 cells show temperature-sensitive growth and temperature-dependent regulation of leucine transport activity. These data suggest that the system L activity of CHO cells is regulated by a dominant-acting element that is defective or absent in the regulatory mutant CHO-C11B6 cell line.

Differential regulation of the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene during oligodendrocyte development.

The two major isoforms of 2',3'-cyclic nucleotide phosphodiesterase (CNP), 48 and 46 kDa, have recently been shown to be produced from a single gene by alternative splicing. In addition, messenger RNA encoding the larger isoform is transcribed from a separate promoter, approximately 1 kb upstream from that encoding the smaller isoform. We have investigated the expression of these two CNP isoforms and have found that they are differentially expressed during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only the mRNA encoding the larger protein is found. At the time of oligodendrocyte differentiation, however, both CNP mRNAs are induced. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation.

Embryonic expression of myelin genes: evidence for a focal source of oligodendrocyte precursors in the ventricular zone of the neural tube.

2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNP) is an abundant protein of myelinating oligodendrocytes. We report that one of the alternatively spliced CNP mRNAs is also expressed in cultured oligodendrocyte progenitor cells. In situ hybridization revealed a thin longitudinal column of CNP-positive cells in the ventral ventricular zone of the embryonic day 14 rat spinal cord, coincident in time and space with cells that express the platelet-derived growth factor alpha receptor, another putative marker of the oligodendrocyte lineage. These data support the hypothesis that the oligodendrocyte lineage originates at a discrete location in the ventral ventricular zone of the embryonic day 14 rat spinal cord. We further report that transcripts encoding the myelin proteolipid protein (PLP/DM-20) are expressed in an unidentified population of neural progenitors in the ventricular zone abutting the floor plate. Our results support the idea that the ventricular zone is a mosaic of specialized progenitor cells.

Down-regulation of the POU transcription factor SCIP is an early event in oligodendrocyte differentiation in vitro.

The POU-domain transcription factor SCIP (also known as Tst-1) has been implicated in the development of Schwann cells, the myelinating cells of the peripheral nervous system (PNS). We have investigated the possibility that SCIP also might play a role in the development of oligodendrocytes, the myelinating cells of the central nervous system (CNS). We purified oligodendrocyte precursors (O-2A progenitors) by immunoselection and cultured them in the presence of platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), which together keep O-2A progenitors proliferating and prevent oligodendrocyte differentiation. Under these culture conditions, O-2A progenitors expressed high levels of SCIP mRNA and protein, and did not express myelin-specific genes. When oligodendrocyte differentiation was initiated by withdrawing the growth factors, SCIP mRNA was rapidly down-regulated, followed by a decline in SCIP protein and the sequential activation of myelin-specific genes. Rapid down-regulation of SCIP mRNA required continued protein synthesis. In O-2A progenitors that were cultured in the presence of PDGF alone, SCIP expression declined to an intermediate level, and low levels of the myelin gene products were induced. Thus, the level of SCIP expression in O-2A progenitors is inversely related to the level of myelin gene expression, suggesting that SCIP may be involved in the developmental switch from proliferation to differentiation in the oligodendrocyte lineage. When O-2A progenitors are cultured in the presence of 10% fetal calf serum, they differentiate into type-2 astrocytes rather than oligodendrocytes. SCIP mRNA was also down-regulated in type-2 astrocytes, which do not express myelin genes, so down-regulation of SCIP seems to be more closely linked to the cessation of cell proliferation per se than the expression of a particular differentiated phenotype.

PDGF receptors in the rat CNS: during late neurogenesis, PDGF alpha-receptor expression appears to be restricted to glial cells of the oligodendrocyte lineage.

Using in situ hybridization, we have visualized cells in the rat central nervous system (CNS) that contain mRNA encoding the platelet-derived growth factor alpha receptor (PDGF-alpha R). After embryonic day 16 (E16), PDGF-alpha R mRNA appears to be expressed by a subset of glial cells, but not by neurons. The temporal and spatial distribution of PDGF-alpha R+ cells, together with 125I-PDGF binding studies on subsets of glial cells in vitro, suggests that PDGF-alpha R may be expressed predominantly, or exclusively, by cells of the oligodendrocyte-type-2 astrocyte (O-2A) lineage. This conclusion is supported by the fact that the numbers of PDGF-alpha R+ cells in developing and adult optic nerves correlate well with independent estimates of the number of O-2A progenitor cells in the nerve at equivalent ages. Small numbers of PDGF-alpha R+ cells are present in the brain at E16, at which time they are found outside the subventricular germinal zones, suggesting that these cells do not express PDGF-alpha R until after, or shortly before they start to migrate away from the subventricular layer towards their final destinations. Reduced numbers of PDGF-alpha R+ cells persist in the adult CNS. PDGF-alpha R is also expressed strongly in the meningeal membranes and choroid plexus, and in the inner limiting membrane of the retina.

Growth factors and transcription factors in oligodendrocyte development.

O-2A progenitor cells, the precursors of oligodendrocytes in the central nervous system (CNS), probably originate in the subventricular germinal zones of the developing CNS, and subsequently migrate away from there to populate the rest of the CNS with oligodendrocytes. We are trying to understand how the O-2A progenitor cells interact with their changing environment as they migrate, and how this influences each stage of their development into mature, myelinating oligodendrocytes. In this article we summarize evidence that platelet-derived growth factor (PDGF) is important for stimulating O-2A progenitor cell proliferation in vivo, and describe our efforts to map the distribution of PDGF and its receptors in the developing rat CNS by in situ hybridization and immunohistochemistry. These studies suggest that, in the CNS, PDGF alpha-receptor subunits may be restricted to O-2A lineage cells that have started to migrate away from the subventricular zones towards their final destinations. Many neurons express the A and/or B chains of PDGF, and astrocytes express the A chain, but it is not yet clear which of these cell types might be the major source of PDGF for O-2A lineage cells in vivo. O-2A progenitor cells can be purified and maintained in a proliferating state in vitro by culturing in the presence of PDGF and bFGF. Under these conditions, the POU transcription factor SCIP/Tst-1 is expressed at a high level; when oligodendrocyte differentiation is initiated by withdrawing the growth factors, SCIP/Tst-1 mRNA is rapidly down-regulated, followed by a decline in SCIP/Tst-1 protein and sequential activation of myelin-specific genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Schwann Cells Secrete a PDGF-like Factor: Evidence for an Autocrine Growth Mechanism involving PDGF.

We have investigated the influence of platelet-derived growth factor (PDGF) in peripheral nervous system gliogenesis using two types of Schwann cell cultures. Short-term Schwann cell cultures grow very slowly, but when maintained in culture for several months the division rate of some cells increases, and cell lines can be established. We show that Schwann cells in both short- and long-term culture possess PDGF receptors and synthesize DNA in response to PDGF. Competitive binding experiments show that Schwann cells express mainly PDGF beta-receptors and respond better to PDGF-BB than to PDGF-AA. Conditioned media from short- and long-term Schwann cell cultures contain PDGF-like mitogenic activity, and anti-PDGF immunoglobin partially inhibits DNA synthesis in long-term Schwann cell cultures. Antibody neutralization experiments and Northern blot analyses both indicate that the predominant PDGF isoform in these cultures is PDGF-BB. PDGF-like activity is also detected in extracts of rat sciatic nerve. Taken together, these results suggest that PDGF-BB may stimulate Schwann cell proliferation in an autocrine manner during normal development.

Characterization of a transport system for anionic amino acids in human fibroblast lysosomes.

L-Aspartate and L-glutamate are transported into human fibroblast lysosomes by a single, low Km, Na(+)-independent transport system, which has been provisionally named lysosomal system d. This system resembles the Na(+)-dependent plasma membrane system chi-AG, although these differences have been observed: (1) lysosomal system d recognizes the D- as well as the L-isomers of both aspartate and glutamate, whereas only for aspartate is the D-isomer recognized by system chi-AG; (2) the anion L-homocysteate is not accepted by system chi-AG, but is an effective inhibitor of lysosomal system d; (3) N-methyl, alpha-methyl, and omega-hydroxamate derivatives of both aspartate and glutamate inhibit lysosomal system d, but only the aspartate derivatives are accepted by system chi-AG; (4) lysosomal system d shows a preference for the substrate amino group in the alpha-position, a preference not seen for system chi-AG. These points imply differences at the two recognition sites with respect to substrate length, size, and rotation, with the lysosomal site generally being the less restrictive.

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids.

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L.

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.

PDGF A chain homodimers drive proliferation of bipotential (O-2A) glial progenitor cells in the developing rat optic nerve.

The bipotential glial progenitor cells (O-2A progenitors), which during development of the rat optic nerve give rise to oligodendrocytes and type 2 astrocytes, are stimulated to divide in culture by platelet-derived growth factor (PDGF), and there is evidence that PDGF is important for development of the O-2A cell lineage in vivo. We have visualized PDGF mRNA in the rat optic nerve by in situ hybridization, and its spatial distribution is compatible with the idea that type 1 astrocytes are the major source of PDGF in the nerve. We can detect mRNA encoding the A chain, but not the B chain of PDGF in the brain and optic nerve, suggesting that the major form of PDGF in the central nervous system is a homodimer of A chains (PDGF-AA). PDGF-AA is a more potent mitogen for O-2A progenitor cells than is PDGF-BB, while the reverse is true for human or rat fibroblasts. Fibroblasts display two types of PDGF receptors, type A receptors which bind to all three dimeric isoforms of PDGF, and type B receptors which bind PDGF-BB and PDGF-AB, but have low affinity for PDGF-AA. Our results suggest that O-2A progenitor cells possess predominantly type A receptors, and proliferate during development in response to PDGF-AA secreted by type 1 astrocytes.