Heike Cappallo-Obermann

Accelerated and safe expansion of human mesenchymal stromal cells in animal serum‐free medium for transplantation and regenerative medicine

Human bone marrow mesenchymal stromal cells (hMSC) are currently investigated for a variety of therapeutic applications. However, most expansion protocols still use fetal calf serum (FCS) as growth factor supplement which is a potential source of undesired xenogeneic pathogens. We established an expansion protocol for hMSC based on the use of GMP‐produced basic medium LP02 supplemented with 5% of platelet lysate (PL) obtained from human thrombocyte concentrates. Compared to FCS‐supplemented culture conditions, we found a significant increase in both colony forming unit‐fibroblast (CFU‐F) as well as cumulative cell numbers after expansion. This accelerated growth is optimized by pooling of at least 10 thrombocyte concentrates. A minimal requirement is the use of 5% of PL with an optimal platelet concentration of 1.5 × 109/ml, and centrifugation of thawed lysate at high speed. Cells expanded by this protocol meet all criteria for mesenchymal stromal cells (MSCs), e.g. plastic adherence, spindle‐shaped morphology, surface marker expression, lack of hematopoietic markers, and differentiation capability into three mesenchymal lineages. MSC at passage 6 were cytogenetically normal and retained their immune‐privileged potential by suppressing allogeneic reaction of T‐cells. Additionally, gene expression profiles show increased mRNA levels of genes involved in cell cycle and DNA replication and downregulation of developmental and differentiation genes, supporting the observation of increased MSC‐expansion in PL‐supplemented medium. In summary, we have established a GMP‐compatible protocol for safe and accelerated expansion of hMSC to be used in cell and tissue therapy. J. Cell. Physiol. 213: 18–26, 2007.

Radiation Rescue: Mesenchymal Stromal Cells Protect from Lethal Irradiation

Background Successful treatment of acute radiation syndromes relies on immediate supportive care. In patients with limited hematopoietic recovery potential, hematopoietic stem cell (HSC) transplantation is the only curative treatment option. Because of time consuming donor search and uncertain outcome we propose MSC treatment as an alternative treatment for severely radiation-affected individuals. Methods and Findings Mouse mesenchymal stromal cells (mMSCs) were expanded from bone marrow, retrovirally labeled with eGFP (bulk cultures) and cloned. Bulk and five selected clonal mMSCs populations were characterized in vitro for their multilineage differentiation potential and phenotype showing no contamination with hematopoietic cells. Lethally irradiated recipients were i.v. transplanted with bulk or clonal mMSCs. We found a long-term survival of recipients with fast hematopoietic recovery after the transplantation of MSCs exclusively without support by HSCs. Quantitative PCR based chimerism analysis detected eGFP-positive donor cells in peripheral blood immediately after injection and in lungs within 24 hours. However, no donor cells in any investigated tissue remained long-term. Despite the rapidly disappearing donor cells, microarray and quantitative RT-PCR gene expression analysis in the bone marrow of MSC-transplanted animals displayed enhanced regenerative features characterized by (i) decreased proinflammatory, ECM formation and adhesion properties and (ii) boosted anti-inflammation, detoxification, cell cycle and anti-oxidative stress control as compared to HSC-transplanted animals. Conclusions Our data revealed that systemically administered MSCs provoke a protective mechanism counteracting the inflammatory events and also supporting detoxification and stress management after radiation exposure. Further our results suggest that MSCs, their release of trophic factors and their HSC-niche modulating activity rescue endogenous hematopoiesis thereby serving as fast and effective first-line treatment to combat radiation-induced hematopoietic failure.

The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART programme

STUDY QUESTION Could the protamine-1 to protamine-2 mRNA ratio serve as a biomarker to estimate the fertilizing capacity of sperm from men taking part in an IVF/ICSI programme? SUMMARY ANSWER The protamine mRNA ratio clearly discriminates between fertile and subfertile men and sperm with a normal protamine mRNA ratio exhibit a higher fertilizing capacity in IVF/ICSI. WHAT IS KNOWN ALREADY Aberrant sperm protamine ratios are associated with male factor infertility and mRNA ratio is comparable with protein ratio (due to transcriptional stop in elongating spermatids). STUDY DESIGN, SIZE, DURATION The study population was drawn from subfertile men, whose female partners participated in IVF or ICSI programmes between September 2010 and February 2012. Normozoospermic healthy volunteers served as controls. Sperm cells were lysed, mRNA extracted, reverse transcribed and subjected to real-time quantitative PCR using specific primer pairs for protamine-1 and protamine-2. Relative protamine-1 and protamine-2 mRNA levels were analysed with the Mann-Whitney U-test (two-tailed). PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative RT-PCR for protamines 1 and 2 has been performed in ejaculates from 32 normozoospermic volunteers (control, University Clinic Giessen, Germany) and 306 patients, whose female partners took part in an IVF (n = 76; University Clinic Hamburg, Germany and Shanghai Jiaotong University, China) or an ICSI (n = 230; University Clinic Munich, Germany and Kinderwunschzentrum Wiesbaden, Germany) programme. MAIN RESULTS AND THE ROLE OF CHANCE The sperm protamine mRNA ratio in normozoospermic men (0.98 ± 0.3) differed significantly from that of ICSI patients (Munich 0.81 ± 0.1; Wiesbaden 0.78 ± 0.2; P < 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0 ± 0.07; Shanghai 1.0 ± 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc > 80%; P = 0.017) and ICSI (65.1% of patients with fc > 70%; P = 0.028). LIMITATIONS, REASONS FOR CAUTION The protamine mRNA ratio in an individual sperm cell used for ICSI may be different from the overall value obtained from a semen aliquot. WIDER IMPLICATIONS OF THE FINDINGS Data are in line with current literature and suggest the protamine mRNA ratio as a diagnostic marker to estimate the fertilizing capacity of sperm. STUDY FUNDING The German Research Foundation (DFG) to K.S., W.W. and A.P. (STE 892/9-2), as well as to A.S. and H.C.O. (SP721/1-3). COMPETING INTEREST(S) None.

Highly purified spermatozoal RNA obtained by a novel method indicates an unusual 28S/18S rRNA ratio and suggests impaired ribosome assembly

Human spermatozoal RNA features special characteristics such as a significantly reduced quantity within spermatozoa compared with somatic cells is described as being devoid of ribosomal RNAs and is difficult to isolate due to a massive excess of genomic DNA in the lysates. Using a novel two-round column-based protocol for human ejaculates delivering highly purified spermatozoal RNA, we uncovered a heterogeneous, but specific banding pattern in microelectrophoresis with 28S ribosomal RNA being indicative for the amount of round cell contamination. Ejaculates with different round cell quantities and density-purified spermatozoa revealed that 18S rRNA but not 28S rRNA is inherent to a pure spermatozoal fraction. Transmission electron microscopy showed monoribosomes and polyribosomes in spermatozoal cytoplasm, while immunohistochemical results suggest the presence of proteins from small and large ribosomal subunits in retained spermatozoal cytoplasm irrespective of 28S rRNA absence.

A One-Step Real-Time Multiplex PCR for Screening Y-Chromosomal Microdeletions without Downstream Amplicon Size Analysis

Backgound Y-chromosomal microdeletions (YCMD) are one of the major genetic causes for non-obstructive azoospermia. Genetic testing for YCMD by multiplex polymerase chain reaction (PCR) is an established method for quick and robust screening of deletions in the AZF regions of the Y-chromosome. Multiplex PCRs have the advantage of including a control gene in every reaction and significantly reducing the number of reactions needed to screen the relevant genomic markers. Principal Findings The widely established “EAA/EMQN best practice guidelines for molecular diagnosis of Y-chromosomal microdeletions (2004)” were used as a basis for designing a real-time multiplex PCR system, in which the YCMD can simply be identified by their melting points. For this reason, some AZF primers were substituted by primers for regions in their genomic proximity, and the ZFX/ZFY control primer was exchanged by the AMELX/AMELY control primer. Furthermore, we substituted the classical SybrGreen I dye by the novel and high-performing DNA-binding dye EvaGreen™ and put substantial effort in titrating the primer combinations in respect to optimal melting peak separation and peak size. Significance With these changes, we were able to develop a platform-independent and robust real-time based multiplex PCR, which makes the need for amplicon identification by electrophoretic sizing expendable. By using an open-source system for real-time PCR analysis, we further demonstrate the applicability of automated melting point and YCMD detection.

Fold-change correction values for testicular somatic transcripts in gene expression studies of human spermatogenesis

STUDY QUESTION What are the reference values for delineating altered somatic cell gene expression from transcript enrichment/dilution in gene expression studies of human spermatogenesis? SUMMARY ANSWER We have designed a crosstable and rule-of-thumb values for different stages of spermatogenic impairment that define the reference cut-off values for altered gene expression in Sertoli and Leydig cells in the context of impaired spermatogenesis. WHAT IS KNOWN ALREADY Morphometrical studies have shown that on the cellular level, impaired spermatogenesis results in a relative enrichment of somatic cell types. However, until now it is not known how this affects transcript levels in gene expression studies. STUDY DESIGN, SIZE DURATION In this study, 31 testis biopsies from men with different stages of spermatogenic impairment (full spermatogenesis, hypospermatogenesis, meiotic arrest, spermatogonial presence, Sertoli cell-only syndrome, complete tubular atrophy) were used to define reference ratios of somatic transcript enrichment/dilution. The reference ratios were validated on an independent test set of 28 samples and on gene expression data from men with Y-chromosomal microdeletions. PARTICIPANTS/MATERIAL, SETTING, METHODS High-quality microarray data were filtered with respect to Sertoli- and Leydig-cell-specific genes. General reference enrichment/dilution factors for these two cell types for all combinations of spermatogenic impairment were calculated using robust permutation statistics. To validate the specificity of the filtered transcripts, we calculated ratios for an independent test set of spermatogenic impairment and for transcriptional data from men with Y-chromosomal microdeletions, and checked the functional enrichment (gene ontology) and cellular localization of the corresponding proteins in a histological database and assessed their correlation with testicular size. MAIN RESULTS AND THE ROLE OF CHANCE Filtering of Sertoli- and Leydig-cell-specific genes resulted in a set of 54 and 332 transcripts, respectively. These were used in defining robust reference dilution/enrichment factors of somatic transcripts for all spermatogenic levels and were compiled in a reference crosstable. Validation on an independent test set showed ratios within 0.5 units of our reference crosstable. Analysis of the resulting transcripts with respect to functional enrichment for Sertoli- and Leydig-cell-specific functions and protein expression, as obtained from an immunohistochemical database, indicated filtering of data sets highly enriched for Sertoli and Leydig cell function. The dilution/enrichment ratios differed significantly when transcripts were interrogated in samples with Y-chromosomal microdeletions, pointing to an overall decreased expression of somatic markers in a genetically altered background. LIMITATIONS, REASONS FOR CAUTION The defined reference ratios might apply with some restrictions in samples that display very heterogeneous histology (e.g. Sertoli cell only with a significant proportion of spermatogenic foci) or when spermatogenic impairment is a consequence of an altered genetic background. WIDER IMPLICATIONS OF THE FINDINGS The reference dilution/enrichment values for somatic testicular transcripts as defined in this study are to be seen as cut-off values for discriminating between simple transcript dilution/enrichment as a consequence of an altered germ cell composition and actual transcriptional regulation. Future studies dealing with transcriptional changes in testicular somatic cells in a background of altered germ cell quantities should consider these correction factors in order to avoid the description of transcriptional changes that are based simply on shifts in somatic cellular quantities. STUDY FUNDING/COMPETING INTEREST(S) Financial support was from grant Sp721/1-3 of the German Research Foundation. There are no competing interests to be declared.

Comment on "Absence of sperm RNA elements correlates with idiopathic male infertility".

The spermatozoal population used for a recent RNA sequencing study may have been contaminated by somatic cell types because of an inefficient sperm purification procedure. The spermatozoal population used for a recent RNA sequencing study may have been contaminated by somatic cell types because of an inefficient sperm purification procedure.

27 The sperm protamine mRNA ratio as a clinical parameter to estimate the fertilizing potential of men taking part in an ART program

main results and the role of chance: The sperm protamine mRNA ratio in normozoospermic men (0.98+ 0.3) differed significantly from that of ICSI patients (Munich 0.81+ 0.1; Wiesbaden 0.78+ 0.2; P , 0.001), while processed samples obtained from IVF patients revealed a normal protamine mRNA ratio (Hamburg 1.0+ 0.07; Shanghai 1.0+ 0.54). Normal protamine mRNA ratios were associated with a significantly higher total motile sperm count and a significantly higher percentage of progressively motile sperm. Sperm with a normal protamine mRNA ratio revealed a higher fertilization capacity (fc) in both IVF (53.6% of patients with fc . 80%; P ¼ 0.017) and ICSI (65.1% of patients with fc . 70%; P ¼ 0.028).