Search

Data suggest that clinical applications of human induced pluripotent stem cells (hiPSCs) will be realized. Nonetheless, clinical applications will require hiPSCs that are free of exogenous DNA and that can be manufactured through Good Manufacturing Practice (GMP). Optimally, derivation of hiPSCs should be rapid and efficient in order to minimize manipulations, reduce potential for accumulation of mutations and minimize financial costs. Previous studies reported the use of modified synthetic mRNAs to reprogram fibroblasts to a pluripotent state. Here, we provide an optimized, fully chemically defined and feeder-free protocol for the derivation of hiPSCs using synthetic mRNAs. The protocol results in derivation of fully reprogrammed hiPSC lines from adult dermal fibroblasts in less than two weeks. The hiPSC lines were successfully tested for their identity, purity, stability and safety at a GMP facility and cryopreserved. To our knowledge, as a proof of principle, these are the first integration-free iPSCs lines that were reproducibly generated through synthetic mRNA reprogramming that could be putatively used for clinical purposes.

Abstract Introduction The reprogramming of a patient’s somatic cells back into induced pluripotent stem cells (iPSCs) holds significant promise for future autologous cellular therapeutics. The continued presence of potentially oncogenic transgenic elements following reprogramming, however, represents a safety concern that should be addressed prior to clinical applications. The polycistronic stem cell cassette (STEMCCA), an excisable lentiviral reprogramming vector, provides, in our hands, the most consistent reprogramming approach that addresses this safety concern. Nevertheless, most viral integrations occur in genes, and exactly how the integration, epigenetic reprogramming, and excision of the STEMCCA reprogramming vector influences those genes and whether these cells still have clinical potential are not yet known. Methods In this study, we used both microarray and sensitive real-time PCR to investigate gene expression changes following both intron-based reprogramming and excision of the STEMCCA cassette during the generation of human iPSCs from adult human dermal fibroblasts. Integration site analysis was conducted using nonrestrictive linear amplification PCR. Transgene-free iPSCs were fully characterized via immunocytochemistry, karyotyping and teratoma formation, and current protocols were implemented for guided differentiation. We also utilized current good manufacturing practice guidelines and manufacturing facilities for conversion of our iPSCs into putative clinical grade conditions. Results We found that a STEMCCA-derived iPSC line that contains a single integration, found to be located in an intronic location in an actively transcribed gene, PRPF39, displays significantly increased expression when compared with post-excised stem cells. STEMCCA excision via Cre recombinase returned basal expression levels of PRPF39. These cells were also shown to have proper splicing patterns and PRPF39 gene sequences. We also fully characterized the post-excision iPSCs, differentiated them into multiple clinically relevant cell types (including oligodendrocytes, hepatocytes, and cardiomyocytes), and converted them to putative clinical-grade conditions using the same approach previously approved by the US Food and Drug Administration for the conversion of human embryonic stem cells from research-grade to clinical-grade status. Conclusion For the first time, these studies provide a proof-of-principle for the generation of fully characterized transgene-free human iPSCs and, in light of the limited availability of current good manufacturing practice cellular manufacturing facilities, highlight an attractive potential mechanism for converting research-grade cell lines into putatively clinical-grade biologics for personalized cellular therapeutics.

Historically, effects of environmental toxicants on human development have been deduced via epidemiological studies because direct experimental analysis has not been possible. However, in recent years, the derivation of human pluripotent stem cells has provided a potential experimental system to directly probe human development. Here, we used human embryonic stem cells (hESCs) to study the effect of environmental toxicants on human germ cell development, with a focus on differentiation of the founding population of primordial germ cells (PGCs), which will go on to form the oocytes of the adult. We demonstrate that human PGC numbers are specifically reduced by exposure to polycyclic aromatic hydrocarbons (PAHs), a group of toxicants common in air pollutants released from gasoline combustion or tobacco smoke. Further, we demonstrate that the adverse effects of PAH exposure are mediated through the aromatic hydrocarbon receptor (AHR) and BAX pathway. This study demonstrates the utility of hESCs as a model system for direct examination of the molecular and genetic pathways of environmental toxicants on human germ cell development.

Several methods allow reprogramming of differentiated somatic cells to embryonic stem cell-like cells. However, the process of reprogramming remains inefficient and the underlying molecular mechanisms are poorly understood. Here, we report the optimization of somatic cell fusion with embryonic stem cells in order to provide an efficient, quantitative assay to screen for factors that facilitate reprogramming. Following optimization, we achieved a reprogramming efficiency 15-590 fold higher than previous protocols. This allowed observation of cellular events during the reprogramming process. Moreover, we demonstrate that overexpression of the Spalt transcription factor, Sall4, which was previously identified as a regulator of embryonic stem cell pluripotency and early mouse development, can enhance reprogramming. The reprogramming activity of Sall4 is independent of an N-terminal domain implicated in recruiting the nucleosome remodeling and deacetylase corepressor complex, a global transcriptional repressor. These results indicate that improvements in reprogramming assays, including fusion assays, may allow the systematic identification and molecular characterization of enhancers of somatic cell reprogramming.

Endogenous retroviruses (ERVs) are remnants of ancient retroviral infections, and comprise nearly 8% of the human genome (1). The most recently acquired human ERV is HERVK (HML-2), which repeatedly infected the primate lineage both before and after the divergence of the human and chimpanzee common ancestor (2, 3). Unlike most other human ERVs, HERVK retained multiple copies of intact open reading frames encoding retroviral proteins (4). However, HERVK is transcriptionally silenced by the host, with the exception of in certain pathological contexts such as germ-cell tumours, melanoma or human immunodeficiency virus (HIV) infection (5-7). Here we demonstrate that DNA hypomethylation at long terminal repeat elements representing the most recent genomic integrations, together with transactivation by OCT4 (also known as POU5F1), synergistically facilitate HERVK expression. Consequently, HERVK is transcribed during normal human embryogenesis, beginning with embryonic genome activation at the eight-cell stage, continuing through the emergence of epiblast cells in preimplantation blastocysts, and ceasing during human embryonic stem cell derivation from blastocyst outgrowths. Remarkably, we detected HERVK viral-like particles and Gag proteins in human blastocysts, indicating that early human development proceeds in the presence of retroviral products. We further show that overexpression of one such product, the HERVK accessory protein Rec, in a pluripotent cell line is sufficient to increase IFITM1 levels on the cell surface and inhibit viral infection, suggesting at least one mechanism through which HERVK can induce viral restriction pathways in early embryonic cells. Moreover, Rec directly binds a subset of cellular RNAs and modulates their ribosome occupancy, indicating that complex interactions between retroviral proteins and host factors can fine-tune pathways of early human development., Given the substantial contribution of transposons to the human genome and their emerging roles in shaping host regulatory networks (8, 9), understanding the dynamic expression and function of these genomic [...]

The leading cause of infertility in men and women is quantitative and qualitative defects in human germ-cell (oocyte and sperm) development. Yet, it has not been possible to examine the [...]

The pathways of gametogenesis encompass elaborate cellular specialization accompanied by precise partitioning of the genome content in order to produce fully matured spermatozoa and oocytes. Transcription factors are an important class of molecules that function in gametogenesis to regulate intrinsic gene expression programs, play essential roles in specifying (or determining) germ cell fate and assist in guiding full maturation of germ cells and maintenance of their populations. Moreover, in order to reinforce or redirect cell fate in vitro, it is transcription factors that are most frequently induced, over-expressed or activated. Many reviews have focused on the molecular development and genetics of gametogenesis, in vivo and in vitro, in model organisms and in humans, including several recent comprehensive reviews: here, we focus specifically on the role of transcription factors. Recent advances in stem cell biology and multi-omic studies have enabled deeper investigation into the unique transcriptional mechanisms of human reproductive development. Moreover, as methods continually improve, in vitro differentiation of germ cells can provide the platform for robust gain- and loss-of-function genetic analyses. These analyses are delineating unique and shared human germ cell transcriptional network components that, together with somatic lineage specifiers and pluripotency transcription factors, function in transitions from pluripotent stem cells to gametes. This grand theme review offers additional insight into human infertility and reproductive disorders that are linked predominantly to defects in the transcription factor networks and thus may potentially contribute to the development of novel treatments for infertility. [ABSTRACT FROM AUTHOR]

Spermatogenesis is the process by which spermatogonial stem ceils divide and differentiate to produce sperm. In vitro sperm production has been difficult to achieve because of the lack of a culture system to maintain viable spermatogonia for long periods of time. Here we report the in vitro generation of spermatocytes and spermatids from telomerase-immortalized mouse type A spermatogonial cells in the presence of stem ceil factor. This differentiation can occur in the absence of supportive ceils. The immortalized spermatogonial cell line may serve as a powerful tool in elucidating the molecular mechanisms of spermatogenesis. Furthermore, through genomic modification and transplantation techniques, this male germ cell line may be used to generate transgenic mice and to develop germ cell gene therapy., Spermatogonia originate from primordial germ cells (PGCs), which are derived from the epiblast by 7.5 days post coitum (dpc) and migrate through the dorsal mesentery and enter the developing fetal [...]

The Deleted in AZoospermia (DAZ) genes encode potential RNA-binding proteins that are expressed exclusively in prenatal and postnatal germ cells and are strong candidates for human fertility factors. Here we report the identification of an additional member of the DAZ gene family, which we have called BOULE. With the identification of this gene, it is clear that the human DAZ gene family contains at least three members: DAZ, a Y-chromosome gene cluster that arose 30-40 million years ago and whose deletion is linked to infertility in men; DAZL, the 'father' of DAZ, a gene that maps to human chromosome 3 and has homologs required for both female and male germ cell development in other organisms; and BOULE, a gene that we propose is the 'grandfather' of DAZ and maps to human chromosome 2. Human and mouse BOULE resemble the invertebrate meiotic regulator Boule, the proposed ortholog of DAZ, in sequence and expression pattern and hence likely perform a similar meiotic function. In contrast, the previously identified human DAZ and DAZL are expressed much earlier than BOULE in prenatal germ stem cells and spermatogonia; DAZL also is expressed in female germ cells. These data suggest that homologs of the DAZ gene family can be grouped into two subfamilies (BOULE and DAZL) and that members of the DAZ family evolved from an ancestral meiotic regulator, Boule, to assume distinct, yet overlapping, functions in germ cell development.

Fertility is a complex process: it is regulated by numerous genetic and environmental factors, and involves coordinate regulation among several organ systems. It is also directly associated with the quantity [...]

Our overall goal is to create a three-dimensional human cell-based testicular model for toxicological and spermatogenesis studies. Methods to purify the major somatic testicular cells, namely Leydig cells (LCs), peritubular myoid cells (PCs) and Sertoli cells (SCs), from rats, mice and guinea pigs have been reported. In humans, the isolation of populations enriched for primary LCs, PCs or SCs also have described. One objective of this study was to determine if populations of cells enriched for all three of these cell types can be isolated from testes of single human donors, and we were successful in doing so from testes of three donors. Testes tissues were enzymatically digested, gravity sedimented and Percoll filtered to isolate populations enriched for LCs, PCs and SCs. LCs and PCs were identified by colorimetric detection of the expression of prototypical enzymes. Division of PCs and SCs in culture has been reported. We observed that primary human LCs could divide in culture by incorporation of 5-ethynyl-2'-deoxyuridine. SCs were identified and their functionality was demonstrated by the formation of tight junctions as shown by the expression of tight junction proteins, increased transepithelial electrical resistance, polarized secretion of biomolecules and inhibition of lucifer yellow penetration. Furthermore, we found that human SC feeder layers could facilitate germ cell progression of human embryonic stem cells (hESCs) by microarray analysis of gene expression. [ABSTRACT FROM AUTHOR]