We have shown that coculture of bone marrow microvascular endothelial cells with hematopoietic progenitor cells results in proliferation and differentiation of megakaryocytes In these long-term cultures, bone marrow microvascular endothelial cell monolayers maintain their cellular integrity in the absence of exogenous endothelial growth factors Because this interaction may involve paracrine secretion of cytokines, we evaluated megakaryocytic cells for secretion of vascular endothelial growth factor (VEGF) Megakaryocytes (CD41a+) were generated by ex vivo expansion of hematopoietic progenitor cells with kit-ligand and thrombopoietin for 10 days and further purified with immunomagnetic microbeads Using reverse transcription–PCR, we showed that megakaryocytic cell lines (Dami, HEL) and purified megakaryocytes expressed mRNA of the three VEGF isoforms (121, 165, and 189 amino acids) Large quantities of VEGF (>1 ng/106 cells/3 days) were detected in the supernatant of Dami cells, ex vivo-generated megakaryocytes, and CD41a+ cells isolated from bone marrow The constitutive secretion of VEGF by CD41a+ cells was stimulated by growth factors of the megakaryocytic lineage (interleukin 3, thrombopoietin) Western blotting of heparin–Sepharose-enriched supernatant mainly detected the isoform VEGF165 In addition, immunohistochemistry showed intracytoplasmic VEGF in polyploid megakaryocytes Thrombin stimulation of megakaryocytes and platelets resulted in rapid release of VEGF within 30 min We conclude that human megakaryocytes produce and secrete VEGF in an inducible manner Within the bone marrow microenvironment, VEGF secreted by megakaryocytes may contribute to the proliferation of endothelial cells VEGF delivered to sites of vascular injury by activated platelets may initiate angiogenesis

https://www.pnas.org/content/pnas/94/2/663.full.pdf

Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets

Aberrant Overexpression of the Wilms Tumor Gene (WT1) in Human Leukemia

The Mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.

Human Bone Marrow Microvascular Endothelial Cells Support Long-Term Proliferation and Differentiation of Myeloid and Megakaryocytic Progenitors

Patients with chronic hepatitis C are more likely to have significant changes in their physical and mental well-being than patients with liver disease of other etiology, and hepatitis C virus (HCV) has been occasionally implicated in diseases of the central nervous system. We analyzed the presence of the HCV negative-strand RNA sequence, which is the viral replicative intermediary, in autopsy brain tissue samples from six HCV-infected patients. Negative-strand HCV RNA was searched for by a strand-specific Tth-based reverse transcriptase PCR, and viral sequences amplified from brain tissue and serum were compared by single-strand conformational polymorphism analysis and direct sequencing. HCV RNA negative strands were detected in brain tissue in three patients. In two of these patients, serum- and brain-derived viral sequences were different and classified as belonging to different genotypes. In one of the latter patients, HCV RNA negative strands were detected in lymph node and, while being different from serum-derived sequences, were identical to those present in the brain. The results of the present study suggest that HCV can replicate in the central nervous system, probably in cells of the macrophage/monocyte lineage.

Search for Hepatitis C Virus Negative-Strand RNA Sequences and Analysis of Viral Sequences in the Central Nervous System: Evidence of Replication

Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction.

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.

Single-cell cDNA-PCR : removal of contaminating genomic DNA from total RNA using immobilized DNase I

To identify possible retrovirus related synovial antigens the immunohistochemistry of rheumatoid synovial tissues was performed. Paraffin-embedded synovial tissues (group 1) from patients with rheumatoid arthritis (RA) were studied with an immunogold technique using monoclonal antibodies against p17 and p24 of the human immunodeficiency virus type I (HIV-I). Immunoreactivity for HIV-I p17 and p24 antigens was detected in 52% and 54% (n=23) of the paraffin-embedded RA specimen, respectively. Immunofluorescence examination of fresh rheumatoid synovial tissues (group 2) revealed reactivity in 60% (n=10) of the cases. RA patients (group 2) whose synovial tissues were reactive by immunofluorescence were seronegative to HIV-I antigens as determined by ELISA and immunoblotting. In one case, reactive tissue processed for immunoelectron microscopy utilizing the immunogold technique, a virus-like structure HIV-I p1. (diameter of 100nm) with an electron-dense core surrounded by a membrane was labeled with antibody to HIV-I p17 reactive electron-dense particles similar in size but lacking membranous structures were also detected in the cytoplasm of 2 different rheumatoid synovial tissues. HIV-I related antigens can be detected in the majority of the rheumatoid synovial tissues in the absence of circulating antibodies to HIV-I. We conclude that HIV-I related antigens might play a role in the pathogenesis of rheumatoid arthritis.

Detection of hiv-i related antigens in rheumatoid synovial tissues

The effect of recombinant human stem cell factor and basic fibroblast growth factor on the in vitro radiosensitivity of CD34+ hematopoietic progenitors from human umbilical cord blood.

Publisher Summary This chapter describes microadapted techniques to isolate messenger RNA (mRNA), or total RNA from only a single cell The isolated mRNA is directly processed for complementary DNA polymerase chain reactions (cDNA PCR), whereas the total RNA is treated with DNase I to remove contaminating genomic DNA The DNase I-treated total RNA is reverse-transcribed into cDNA and then used for cDNA-PCR analysis To determine the mRNA phenotype of cells by cDNA-PCR, two oligonucleotide primers that can recognize the (+)-strand and the (–)-strand of the target gene are required The target sequence is amplified exponentially by repeated cycles of denaturation, primer annealing, and primer extension in the presence of thermostable DNA polymerase The analysis of the mRNA phenotype of a small number of cells by cDNA-PCR requires the isolation of sufficient quantities of RNA This approach is hampered by the fact that standard protocols for the isolation of RNA cannot be applied Therefore, techniques scaled down for the isolation of small amounts of RNA, such as the microadapted guanidine thiocyanate (GuSCN)/CsCl gradient centrifugation, and protocols for the direct isolation of poly(A) RNA from cellular lysates have been developed

Benedikt L. Ziegler

Papers

Phenotype analysis of hematopoietic CD34+ cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction.

Single-cell cDNA-PCR : removal of contaminating genomic DNA from total RNA using immobilized DNase I

Detection of hiv-i related antigens in rheumatoid synovial tissues

The effect of recombinant human stem cell factor and basic fibroblast growth factor on the in vitro radiosensitivity of CD34+ hematopoietic progenitors from human umbilical cord blood.

[5] Single-cell cDNA-PCR