Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium.

BACKGROUND: The production of human platelets from embryonic stem cells in a defined culture system is a prerequisite for the generation of platelets for therapeutic use. As an important step towards this goal, we report the differentiation of human embryonic stem cells (hESCs) towards the megakaryocyte (Mk) lineage using a 'spin embryoid body' method in serum-free differentiation medium. METHODOLOGY AND PRINCIPAL FINDINGS: Immunophenotypic analyses of differentiating hESC identified a subpopulation of cells expressing high levels of CD41a that expressed other markers associated with the Mk lineage, including CD110, CD42b and CD61. Differentiated cells were sorted on the basis of their expression of CD41a, CD34 and CD45 and assessed for Mk colony formation, expression of myeloid and Mk genes and ability to endoreplicate DNA. In a collagen-based colony assay, the CD41a⁺ cells sorted from these differentiation cultures produced 100-800 Mk progenitors at day 13 and 25-160 Mk progenitors at day 20 of differentiation per 100,000 cells assayed. Differentiated Mk cells produced platelet-like particles which expressed CD42b and were activated by ADP, similar to platelets generated from precursors in cord blood. These studies were complemented by real time PCR analyses showing that subsets of cells enriched for CD41a⁺ Mk precursors expressed high levels of Mk associated genes such as PF4 and MPL. Conversely, high levels of myeloid and erythroid related transcripts, such as GATA1, TAL1/SCL and PU.1, were detected in sorted fractions containing CD34⁺ and CD45⁺ cells. CONCLUSIONS: We describe a serum- and feeder-free culture system that enabled the generation of Mk progenitors from human embryonic stem cells. These cells formed colonies that included differentiated Mks that fragmented to form platelet-like particles. This protocol represents an important step towards the generation of human platelets for therapeutic use.

Supersensitive fluorescent semen analysis: validation on azoospermic and oligozoospermic samples.

OBJECTIVE: To compare the clinical utility of a supersensitive fluorescent semen analysis (SSSA) procedure published by Cooper et al. (2006) with a conventional World Health Organization (WHO)-based semen analysis technique in males with severe oligozoospermia or azoospermia who are undergoing fertility assessment. DESIGN: Prospective single-center study. SETTING: IVF clinic. PATIENT(S): Patients attending an infertility clinic for semen analysis. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Presence of spermatozoa in the ejaculate. RESULT(S): Semen samples from 100 men were analyzed using conventional WHO 4th Edition semen analysis and determined to be either severely oligozoospermic or azoospermic (reported lower limit of detection of 0.1 million sperm/mL). An aliquot of the same unprocessed sample was also analyzed using the SSSA protocol (reported lower limit of detection of approximately 8000 sperm/mL). The SSSA method confirmed the results of conventional semen analysis in 77% of cases. In 22% of cases, sperm were identified only using SSSA. Overall, SSSA was capable of identifying the presence of sperm in significantly more samples than conventional semen analysis. CONCLUSION(S): The reliable differentiation of extreme oligospermia from azoospermia has profound implications in fertility management. This paper provides the first data comparing sperm detection rates using SSSA or conventional WHO-based approaches in extreme oligozoospermic and azoospermic men in an IVF setting. Results indicate that approximately one in four men classified as azoospermic by conventional semen analysis may actually have sperm present. The improved sensitivity of the SSSA technique may be of significant benefit to patients, particularly in fertility and assisted reproductive technique decision making.

A QF-PCR system to detect chromosome 13 aneuploidy from as few as ten cells.

Complete and partial trisomies of chromosome 13 are characterized by abnormal fetal development and birth defects. Despite the severe abnormalities associated with trisomy 13, some couples elect not to undergo invasive prenatal diagnosis (PND) due to the 0.5%-1.0% risk of pregnancy loss. As a result, current studies are focusing on refining non-invasive prenatal diagnostic techniques, such as screening fetal cells isolated from maternal blood or cervical smears. As these techniques only provide a limited number of cells for analysis, any progress in this field depends on the development of a highly sensitive genetic screening strategy. We have developed a quantitative fluorescent PCR (QF-PCR) system capable of detecting chromosome 13 aneuploidy from as few as 10 cells. The system was further validated by screening 13 amniocyte samples, three of which had been diagnosed by FISH as having chromosomal abnormalities involving chromosome 13. In all cases, the QF-PCR results were concordant with those obtained using FISH. The high reliability (99%) and accuracy (96%) of the QF-PCR system at the 10 cell level makes this technique ideal for use in non-invasive PND.