Biological therapy for non-obstructive azoospermia.

INTRODUCTION: Most male patients with non-obstructive azoospermia (NOA) have no therapeutic options outside of assisted reproductive techniques to conceive a biological child. If mature sperm cannot be obtained from the testes, these patients must rely on options of donor sperm or adoption. Several techniques are in the experimental stage to provide this patient population alternatives for conceiving. AREAS COVERED: This review discusses three of the experimental techniques for restoring fertility in men with NOA: spermatogonial stem cell transplantation, the use of adult and embryonic stem cells to develop mature gametes and gene therapy. After this discussion, the authors give their expert opinion and provide the reader with their perspectives for the future. EXPERT OPINION: Several limitations, both technical and ethical, exist for spermatogonial stem cell transplantation, the use of stem cells and gene therapy. Well-defined reproducible protocols are necessary. Furthermore, several technical barriers exist for all protocols. And while success has been achieved in animal models, future research is still required in human models.

Human pluripotent stem cell strategies for age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the Western world and is increasing exponentially as the population ages. Despite enormous worldwide efforts, the earliest pathogenic pathways involved in AMD are still not fully understood. It is essential to develop research tools for effective modeling of AMD pathogenesis and for subsequent drug discovery and cell or molecular therapies. This review will focus on the current progress in human pluripotent stem cells for understanding and treating AMD.

Pluripotency and targeted reprogramming: strategies, disease modeling and drug screening.

Pluripotent stem cell research has developed over the last fifty years from the study of embryonic development to a multifaceted discipline that encompasses development, epigenetics, reprogramming, cell therapy, disease modeling and chemical and drug screening. The idea of patient-specific therapies and disease modeling using human pluripotent stem cells has been the theoretical golden-egg of the field since the generation of human embryonic stem cells. With the advent of induced pluripotent stem cells (PSCs), the ability to generate patient-specific cells for therapeutic use, to model disease progression and to test drugs on disease relevant cells moved a large step closer to reality. While there still is a long way to go before the results of PSC research is found in the clinic or in the pharmacy, recent developments have demonstrated that it is possible to generate patient-specific pluripotent cells which can differentiate into disease relevant cell types, are amenable to gene correction, can phenocopy molecular and functional disease characteristics, at least in vitro, and can be used to validate the efficacy of therapeutic compounds. This review will cover recent developments in the generation and manipulation of pluripotent stem cells with a focus on the use of pluripotent stem cells for disease modeling and therapeutic drug screening. In addition, the latest developments in somatic cell reprogramming will also be discussed.

Human mid-trimester amniotic fluid stem cells cultured under embryonic stem cell conditions with valproic acid acquire pluripotent characteristics.

Human mid-trimester amniotic fluid stem cells (AFSC) have promising applications in regenerative medicine, being broadly multipotent with an intermediate phenotype between embryonic (ES) and mesenchymal stem cells (MSC). Despite this propluripotent phenotype, AFSC are usually cultured in adherence in a serum-based expansion medium, and how expansion in conditions sustaining pluripotency might affect their phenotype remains unknown. We recently showed that early AFSC from first trimester amniotic fluid, which endogenously express Sox2 and Klf4, can be reprogrammed to pluripotency without viral vectors using the histone deacetylase inhibitor valproic acid (VPA). Here, we show that mid-trimester AFSC cultured under MSC conditions contained a subset of cells endogenously expressing telomerase, CD24, OCT4, C-MYC, and SSEA4, but low/null levels of SOX2, NANOG, KLF4, SSEA3, TRA-1-60, and TRA-1-81, with cells unable to form embryoid bodies (EBs) or teratomas. In contrast, AFSC cultured under human ESC conditions were smaller in size, grew faster, formed colonies, upregulated OCT4 and C-MYC, and expressed KLF4 and SOX2, but not NANOG, SSEA3, TRA-1-60, and TRA-1-81. Supplementation with VPA for 5 days further upregulated OCT4, KLF4, and SOX2, and induced expression of NANOG, SSEA3, TRA-1-60, and TRA-1-81, with cells now able to form EBs and teratomas. We conclude that human mid-trimester AFSC, which may be isolated autologously during pregnancy without ethics restriction, can acquire pluripotent characteristics without the use of ectopic factors. Our data suggest that this medium-dependant approach to pluripotent mid-trimester AFSC reflects true reprogramming and not the selection of prepluripotent cells.

Cerebral palsy: the whys and hows.

The descriptive term of cerebral palsy encompasses the largest group of childhood movement disorders. Severity and pattern of clinical involvement varies widely dependent on the area of the central nervous system compromised. A multidisciplinary team approach is vital for all the aspects of management to improve function and minimise disability. From a medical viewpoint, there are two pronged approaches. First a focus on developmental and clinical comorbidities such as communication, behaviour, epilepsy, feeding problems, gastro-oesophageal reflux and infections; and second on specifics of muscle tone, motor control and posture. With regards to the latter, there is an increasing number of available treatments including oral antispasticity and antidystonic medications, injectable botulinum toxin, multilevel orthopaedic and neurosurgical options and a variety of complementary and alternative therapies.

Primary cells and stem cells in drug discovery: emerging tools for high-throughput screening.

Many drug discovery screening programs employ immortalized cells, recombinantly engineered to express a defined molecular target. Several technologies are now emerging that render it feasible to employ more physiologically, and clinically relevant, cell phenotypes. Consequently, numerous approaches use primary cells, which retain many functions seen in vivo, as well as endogenously expressing the target of interest. Furthermore, stem cells, of either embryonic or adult origin, as well as those derived from differentiated cells, are now finding a place in drug discovery. Collectively, these cells are expanding the utility of authentic human cells, either as screening tools or as therapeutics, as well as providing cells derived directly from patients. Nonetheless, the growing use of phenotypically relevant cells (including primary cells or stem cells) is not without technical difficulties, particularly when their envisioned use lies in high-throughput screening (HTS) protocols. In particular, the limited availability of homogeneous primary or stem cell populations for HTS mandates that novel technologies be developed to accelerate their adoption. These technologies include detection of responses with very few cells as well as protocols to generate cell lines in abundant, homogeneous populations. In parallel, the growing use of changes in cell phenotype as the assay readout is driving greater use of high-throughput imaging techniques in screening. Taken together, the greater availability of novel primary and stem cell phenotypes as well as new detection technologies is heralding a new era of cellular screening. This convergence offers unique opportunities to identify drug candidates for disorders at which few therapeutics are presently available.

Emerging concepts in neural stem cell research: autologous repair and cell-based disease modelling.

The increasing availability of human pluripotent stem cells provides new prospects for neural-replacement strategies and disease-related basic research. With almost unlimited potential for self-renewal, the use of human embryonic stem cells (ESCs) bypasses the restricted supply and expandability of primary cells that has been a major bottleneck in previous neural transplantation approaches. Translation of developmental patterning and cell-type specification techniques to human ESC cultures enables in vitro generation of various neuronal and glial cell types. The derivation of stably proliferating neural stem cells from human ESCs further facilitates standardisation and circumvents the problem of batch-to-batch variations commonly encountered in "run-through" protocols, which promote terminal differentiation of pluripotent stem cells into somatic cell types without defined intermediate precursor stages. The advent of cell reprogramming offers an opportunity to translate these advances to induced pluripotent stem cells, thereby enabling the generation of neurons and glia from individual patients. Eventually, reprogramming could provide a supply of autologous neural cells for transplantation, and could lead to the establishment of cellular model systems of neurological diseases.

Generation of mouse induced pluripotent stem cells without viral vectors.

Induced pluripotent stem (iPS) cells have been generated from mouse and human somatic cells by introducing Oct3/4 and Sox2 with either Klf4 and c-Myc or Nanog and Lin28 using retroviruses or lentiviruses. Patient-specific iPS cells could be useful in drug discovery and regenerative medicine. However, viral integration into the host genome increases the risk of tumorigenicity. Here, we report the generation of mouse iPS cells without viral vectors. Repeated transfection of two expression plasmids, one containing the complementary DNAs (cDNAs) of Oct3/4, Sox2, and Klf4 and the other containing the c-Myc cDNA, into mouse embryonic fibroblasts resulted in iPS cells without evidence of plasmid integration, which produced teratomas when transplanted into mice and contributed to adult chimeras. The production of virus-free iPS cells, albeit from embryonic fibroblasts, addresses a critical safety concern for potential use of iPS cells in regenerative medicine.