• Human embryonic stem cells
• Pluripotent stem cells
• Retinal stem cell
• Retinal tissue transplant
• Retinal transplantation

• culture medium
• embryonic stem cells
• genome integrity
• lipids
• pluripotency

• Genomic Instability
• Humans
• Animals
• Lipids/chemistry
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/metabolism
• Induced Pluripotent Stem Cells/metabolism
• Induced Pluripotent Stem Cells/cytology
• Cell Differentiation
• Pluripotent Stem Cells/metabolism
• Pluripotent Stem Cells/cytology
• Lipid Metabolism

• R01 GM129380 NIGMS NIH HHS
• R21 OD031973 NIH HHS
• National Institute of Health
• New York State Stem Cell Science Program
• NYSTEM

• 20q11.21
• cancer
• clinical translation
• genetic integrity
• human pluripotent stem cells

• Humans
• Pluripotent Stem Cells
• Mutation
• Cell Transformation, Neoplastic/genetics
• Cell Transformation, Neoplastic/pathology
• Aneuploidy
• Cell Differentiation/genetics

• DNA methylation
• chromatin accessibility
• epigenome
• histone modifications
• stem cell-based embryo models

• Humans
• DNA Methylation
• Embryo, Mammalian/metabolism
• Embryonic Development/genetics
• Epigenesis, Genetic

• POFUT1
• head and neck squamous cell carcinoma
• oncogene
• perineural invasion

• Humans
• Squamous Cell Carcinoma of Head and Neck/genetics
• Carcinoma, Squamous Cell/genetics
• Carcinoma, Squamous Cell/pathology
• Head and Neck Neoplasms/genetics
• Phenotype
• Carcinogenesis
• Cell Line, Tumor
• Cell Proliferation/genetics

• 118Z767 Scientific and Technological Research Council of Turkey

• head and neck squamous cell carcinoma
• miR-1825
• oncogene

• 118Z767 Scientific and Technological Research Council of Turkey
• 121Z004 Scientific and Technological Research Council of Turkey

• exosomes
• hypoxia
• miR-1825
• microRNA
• oral squamous cell carcinoma

• Humans
• Carcinoma, Squamous Cell/pathology
• Cell Line, Tumor
• Cell Proliferation
• Exosomes/genetics
• Exosomes/metabolism
• Exosomes/pathology
• Head and Neck Neoplasms/pathology
• Human Umbilical Vein Endothelial Cells/metabolism
• Human Umbilical Vein Endothelial Cells/pathology
• Hypoxia/metabolism
• Hypoxia/pathology
• MicroRNAs/genetics
• MicroRNAs/metabolism
• Mouth Neoplasms/pathology
• Squamous Cell Carcinoma of Head and Neck/pathology
• TOR Serine-Threonine Kinases/metabolism

• Cancer stem cell
• Hepatitis B virus
• Hepatitis C virus
• Hepatocellular carcinoma
• Liver cirrhosis
• miRNA

Millions of people around the world suffer from infertility, with the number of infertile couples and individuals increasing every year. Assisted reproductive technologies (ART) have been widely developed in recent years; however, some patients are unable to benefit from these technologies due to their lack of functional germ cells. Therefore, the development of alternative methods seems necessary. One of these methods is to create artificial oocytes. Oocytes can be generated in vitro from the ovary, fetal gonad, germline stem cells (GSCs), ovarian stem cells, or pluripotent stem cells (PSCs). This approach has raised new hopes in both basic research and medical applications. In this article, we looked at the principle of oocyte development, the landmark studies that enhanced our understanding of the cellular and molecular mechanisms that govern oogenesis in vivo, as well as the mechanisms underlying in vitro generation of functional oocytes from different sources of mouse and human stem cells. In addition, we introduced next-generation ART using somatic cells with artificial oocytes. Finally, we provided an overview of the reproductive application of in vitro oogenesis and its use in human fertility.

• artificial oocyte
• assisted reproductive technologies
• haploidization of somatic chromosomes
• oogenesis
• stem cells

• cell therapy
• clinical translation
• standard
• stem cell

• Cell- and Tissue-Based Therapy
• Humans
• Pluripotent Stem Cells
• Reference Standards
• Regenerative Medicine
• Stem Cell Research

• ALDH
• CD133
• LGR5
• Organoids
• PROCR
• adult stem cells
• beta cell
• beta cell regeneration
• diabetes
• pancreas
• progenitor cells
• stem cell therapy
• transplantation

• Cell Differentiation
• Diabetes Mellitus/therapy
• Humans
• Organoids
• Pancreas
• Pluripotent Stem Cells

Retinitis pigmentosa is the most common hereditary retinal disease. Dietary supplements, neuroprotective agents, cytokines, and lately, prosthetic devices, gene therapy, and optogenetics have been employed to slow down the retinal degeneration or improve light perception. Completing retinal circuitry by transplanting photoreceptors has always been an appealing idea in retinitis pigmentosa. Recent developments in stem cell technology, retinal imaging techniques, tissue engineering, and transplantation techniques have brought us closer to accomplish this goal. The eye is an ideal organ for cell transplantation due to a low number of cells required to restore vision, availability of safe surgical and imaging techniques to transplant and track the cells in vivo, and partial immune privilege provided by the subretinal space. Human embryonic stem cells, induced pluripotential stem cells, and especially retinal organoids provide an adequate number of cells at a desired developmental stage which may maximize integration of the graft to host retina. However, stem cells must be manufactured under strict good manufacturing practice protocols due to known tumorigenicity as well as possible genetic and epigenetic stabilities that may pose a danger to the recipient. Immune compatibility of stem cells still stands as a problem for their widespread use for retinitis pigmentosa. Transplantation of stem cells from different sources revealed that some of the transplanted cells may not integrate the host retina but slow down the retinal degeneration through paracrine mechanisms. Discovery of a similar paracrine mechanism has recently opened a new therapeutic path for reversing the cone dormancy and restoring the sight in retinitis pigmentosa.

The genomic stability of stem cells to be used in cell therapy and other clinical applications is absolutely critical. In this regard, the relationship between in vitro expansion and the chromosomal instability (CIN), especially in human amniotic fluid cells (hAFCs) has not yet been completely elucidated.

To investigate the CIN of hAFCs in primary and long-term cultures and two different culture mediums.

After completing prenatal genetic diagnoses (PND) using karyotype technique and chromosomal analysis, a total of 15 samples of hAFCs from 650 samples were randomly selected and cultured in two different mediums as AmnioMAX II and DMEM. Then, proliferative cells were fixed on the slide to be used in standard chromosome G-banding analysis. Also, the senescent cells were screened for aneuploidy considering 8 chromosomes by FISH technique using two probe sets including PID I (X-13-18-21) & PID II (Y-15-16-22).

Karyotype and interphase fluorescence in situ hybridization (iFISH) results from 650 patients who were referred for prenatal genetic diagnosis showed that only 6 out of them had culture- derived CIN as polyploidy, including mosaic diploid-triploid and diploid-tetraploid. Moreover, the investigation of aneuploidies in senesced hAFCs demonstrated the rate of total chromosomal abnormalities as 4.3% and 9.9% in AmnioMAX- and DMEM-cultured hAFCs, respectively.

hAFCs showed a low rate of CIN in two AmnioMAX II and DMEM mediums and also in the proliferative and senescent phases. Therefore, they could be considered as an attractive stem cell source with therapeutic potential in regenerative medicine.

• Amniocentesis
• Chromosomal instability
• Pseudomosaicism
• Replicative senescence.
• Human amniotic fluid cells

• TGF‐β signaling pathway
• chimera formation
• human naïve pluripotency
• nuclear receptors

• Animals
• Blastocyst
• Cell Differentiation
• Cell Line
• Humans
• Mice
• Pluripotent Stem Cells
• Receptors, Cytoplasmic and Nuclear/genetics
• Receptors, Retinoic Acid
• Retinoic Acid Receptor gamma

• Royan Institute
• Iranian Council of Stem Cell Research and Technology (ICSCR)
• Iran National Science Foundation (INSF)
• SFB TRR81 TP A02 Deutsche Forschungsgemeinschaft (DFG)
• SFB 1213 Deutsche Forschungsgemeinschaft (DFG)
• TP A02 Deutsche Forschungsgemeinschaft (DFG)
• B02 Deutsche Forschungsgemeinschaft (DFG)
• European Research Area Network on Cardiovascular Diseases
• Royan Institute
• Iranian Council of Stem Cell Research and Technology (ICSCR)
• Iran National Science Foundation (INSF)
• Deutsche Forschungsgemeinschaft (DFG)

Embryonic stem cells (ESCs) derived from somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) are promising tools for meeting the personalized requirements of regenerative medicine. However, some obstacles need to be overcome before clinical trials can be undertaken. First, donor cells vary, and the reprogramming procedures are diverse, so standardization is a great obstacle regarding SCNT and iPSCs. Second, somatic cells derived from a patient may carry mitochondrial DNA mutations and exhibit telomere instability with aging or disease, and SCNT-ESCs and iPSCs retain the epigenetic memory or epigenetic modification errors. Third, reprogramming efficiency has remained low. Therefore, in addition to improving their success rate, other alternatives for producing ESCs should be explored. Producing androgenetic diploid embryos could be an outstanding strategy; androgenic diploid embryos are produced through double sperm cloning (DSC), in which two capacitated sperms (XY or XX, sorted by flow cytometer) are injected into a denucleated oocyte by intracytoplasmic sperm injection (ICSI) to reconstruct embryo and derive DSC-ESCs. This process could avoid some potential issues, such as mitochondrial interference, telomere shortening, and somatic epigenetic memory, all of which accompany somatic donor cells. Oocytes are naturally activated by sperm, which is unlike the artificial activation that occurs in SCNT. The procedure is simple and practical and can be easily standardized. In addition, DSC-ESCs can overcome ethical concerns and resolve immunological response matching with sperm providers. Certainly, some challenges must be faced regarding imprinted genes, epigenetics, X chromosome inactivation, and dosage compensation. In mice, DSC-ESCs have been produced and have shown excellent differentiation ability. Therefore, the many advantages of DSC make the study of this process worthwhile for regenerative medicine and animal breeding.

• Double sperm cloning
• Embryonic stem cell
• Regenerative medicine
• Reprogramming

Gain of 20q11.21 is one of the most common recurrent genomic aberrations in human pluripotent stem cells. Although it is known that overexpression of the antiapoptotic gene Bcl-xL confers a survival advantage to the abnormal cells, their differentiation capacity has not been fully investigated. RNA sequencing of mutant and control hESC lines, and a line transgenically overexpressing Bcl-xL, shows that overexpression of Bcl-xL is sufficient to cause most transcriptional changes induced by the gain of 20q11.21. Moreover, the differentially expressed genes in mutant and Bcl-xL overexpressing lines are enriched for genes involved in TGF-β- and SMAD-mediated signaling, and neuron differentiation. Finally, we show that this altered signaling has a dramatic negative effect on neuroectodermal differentiation, while the cells maintain their ability to differentiate to mesendoderm derivatives. These findings stress the importance of thorough genetic testing of the lines before their use in research or the clinic.

•

Bcl-xL overexpression drives the transcriptomic profile of 20q11.21 mutant lines

• 20q11.21
• chromosomal abnormalities
• genome instability
• human embryonic stem cells
• human pluripotent stem cells
• impaired differentiation
• neuroectoderm

• Cell Differentiation/genetics
• Chromosome Aberrations
• Chromosomes, Human, Pair 20/chemistry
• Chromosomes, Human, Pair 20/genetics
• DNA-Binding Proteins/genetics
• Down-Regulation
• Gene Amplification
• Humans
• Neural Plate/cytology
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism
• Sequence Analysis, RNA
• Signal Transduction
• Smad Proteins/genetics
• Smad Proteins/metabolism
• Transcription Factors/genetics
• Transforming Growth Factor beta/genetics
• Transforming Growth Factor beta/metabolism
• bcl-X Protein/genetics
• bcl-X Protein/metabolism

• adverse event
• clinical translation
• evolution
• genetic stability
• pluripotent stem-cell
• safety
• stem cell
• stem cell therapy
• stem-cell research

How did the field of stem cell research develop in the years following the derivation of the first human embryonic stem cell (hESC) line?

Supported by the increasing number of clinical trials to date, significant technological advances in the past two decades have brought us ever closer to clinical therapies derived from pluripotent cells.

Since their discovery 20 years ago, the use of human pluripotent stem cells has progressed tremendously from bench to bedside. Here, we provide a concise review of the main keystones of this journey and focus on ongoing clinical trials, while indicating the most relevant future research directions.

This is a historical narrative, including relevant publications in the field of pluripotent stem cells (PSC) derivation and differentiation, recounted both through scholarly research of published evidence and interviews of six pioneers who participated in some of the most relevant discoveries in the field.

The authors all contributed by researching the literature and agreed upon body of works. Portions of the interviews of the field pioneers have been integrated into the review and have also been included in full for advanced reader interest.

The stem cell field is ever expanding. We find that in the 20 years since the derivation of the first hESC lines, several relevant developments have shaped the pluripotent cell field, from the discovery of different states of pluripotency, the derivation of induced PSC, the refinement of differentiation protocols with several clinical trials underway, as well as the recent development of organoids. The challenge for the years to come will be to validate and refine PSCs for clinical use, from the production of highly defined cell populations in clinical grade conditions to the possibility of creating replacement organoids for functional, if not anatomical, function restoration.

This is a non-systematic review of current literature. Some references may have escaped the experts’ analysis due to the exceedingly diverse nature of the field. As the field of regenerative medicine is rapidly advancing, some of the most recent developments may have not been captured entirely.

The multi-disciplinary nature and tremendous potential of the stem cell field has important implications for basic as well as translational research. Recounting these activities will serve to provide an in-depth overview of the field, fostering a further understanding of human stem cell and developmental biology. The comprehensive overview of clinical trials and expert opinions included in this narrative may serve as a valuable scientific resource, supporting future efforts in translational approaches.

ESHRE provided funding for the authors’ on-site meeting and discussion during the preparation of this manuscript. S.M.C.S.L. is funded by the European Research Council Consolidator (ERC-CoG-725722-OVOGROWTH). M.P. is supported by the Special Research Fund, Bijzonder Onderzoeksfonds (BOF01D08114). M.G. is supported by the Methusalem grant of Vrije Universiteit Brussel, in the name of Prof. Karen Sermon and by Innovation by Science and Technology in Flanders (IWT, Project Number: 150042). A.V. and B.A. are supported by the Plataforma de Proteomica, Genotipado y Líneas Celulares (PT1770019/0015) (PRB3), Instituto de Salud Carlos III. Research grant to B.H. by the Research Foundation—Flanders (FWO) (FWO.KAN.2016.0005.01 and FWO.Project G051516N). There are no conflicts of interest to declare.

Not applicable.

ESHRE Pages are not externally peer reviewed. This article has been approved by the Executive Committee of ESHRE.

• clinical trials
• differentiation
• human embryonic stem cell (hESC)
• human-induced pluripotent stem cells (hiPSC)
• pluripotency
• regenerative medicine

• SOX2
• non-functional pituitary adenoma
• pituitary adenomas
• secreting adenoma

• Female
• Gene Expression Regulation/genetics
• Humans
• Immunohistochemistry
• Male
• Pituitary Gland/metabolism
• Pituitary Gland/pathology
• Pituitary Neoplasms/genetics
• Pituitary Neoplasms/pathology
• SOXB1 Transcription Factors/genetics

• Epigenetics
• Mitochondria
• Mitochondrial DNA
• Stem cell

• genome instability
• mitochondria
• mosaicism
• mtDNA
• pluripotent stem cells

• Alleles
• Cell Culture Techniques
• Cell Differentiation/genetics
• Chromosome Aberrations
• Clonal Evolution/genetics
• DNA, Mitochondrial
• Fibroblasts/metabolism
• Gene Expression Profiling
• Genetic Heterogeneity
• Genetic Variation
• Genomic Instability
• Genotype
• Humans
• Mosaicism
• Mutagenesis
• Pluripotent Stem Cells/metabolism

• Biotechnology
• Cell- and Tissue-Based Therapy
• Humans
• Immunotherapy

• R01 EB012521 NIBIB NIH HHS
• T32 EB016652 NIBIB NIH HHS

• CRISPR-Cas9
• CRISPR-Cas9 nickase
• human pluripotent stem cells
• psychiatric disease
• stem cell genomic stability
• stem cell-derived human neurons
• variant calling

• Biomarkers
• CRISPR-Cas Systems
• Cell Differentiation/genetics
• Cell Line
• Gene Editing
• Gene Expression
• Gene Targeting
• Genes, Reporter
• Genomic Instability
• Humans
• INDEL Mutation
• Mental Disorders/etiology
• Mental Disorders/metabolism
• Mental Disorders/psychology
• Neurons/cytology
• Neurons/metabolism
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism
• Workflow

• CpG island methylator phenotype
• chromosomal instability
• colorectal cancer
• early diagnosis
• microsatellite instability
• molecular characteristics
• targeted therapy

• Fuzzy inheritance
• Genome chaos
• Genome re-organization
• Genome theory
• Heterogeneity
• Karyotype coding
• Parts inheritance
• System inheritance
• Systems biology

• X chromosome inactivation
• differentiation
• embryonic stem cells
• epigenetics
• human
• in vitro modelling
• induced pluripotent stem cells
• preimplantation development
• primordial germ cells

• clinical translation
• differentiation propensity
• epigenetic variance
• genetic abnormalities
• genetic diversity
• human embryonic stem cells
• human induced pluripotent stem cells
• mitochondria
• predictive markers

• Cell culture
• Differentiation
• Embryonic stem cells
• Technology
• Tissue engineering

Corneal transplantation is the only treatment available to restore vision for individuals with blindness due to corneal endothelial dysfunction. However, severe shortage of available donor corneas remains a global challenge. Functional regulatory compliant tissue-engineered corneal endothelial graft substitute can alleviate this reliance on cadaveric corneal graft material. Here, isolated primary human corneal endothelial cells (CEnCs) propagated using a dual media approach refined towards regulatory compliance showed expression of markers indicative of the human corneal endothelium, and can be tissue-engineered onto thin corneal stromal carriers. Both cellular function and clinical adaptability was demonstrated in a pre-clinical rabbit model of bullous keratopathy using a tissue-engineered endothelial keratoplasty (TE-EK) approach, adapted from routine endothelial keratoplasty procedure for corneal transplantation in human patients. Cornea thickness of rabbits receiving TE-EK graft gradually reduced over the first two weeks, and completely recovered to a thickness of approximately 400 µm by the third week of transplantation, whereas corneas of control rabbits remained significantly thicker over 1,000 µm (p < 0.05) throughout the course of the study. This study showed convincing evidence of the adaptability of the propagated CEnCs and their functionality via a TE-EK approach, which holds great promises in translating the use of cultured CEnCs into the clinic.

• Chromosomal aberrations
• Copy number variations
• Induced pluripotent stem cells
• Insertions and deletions
• Karyotype abnormalities
• Single nucleotide variants
• iPS cells

• Animals
• Cellular Reprogramming/genetics
• Cellular Reprogramming/physiology
• Embryonic Stem Cells/physiology
• Genomic Instability/physiology
• Pluripotent Stem Cells/physiology
• Stem Cells/physiology

• Epigenetics
• Epigenomics
• Genome editing
• Neurological disorders
• Neuronal differentiation
• Stem cell reprogramming
• Stem cell technologies
• iPS cell based disease modelling

Emerging evidences proposed that microRNAs are associated with regulation of distinct physio-pathological processes including development of normal stem cells and carcinogenesis. In this study we aimed to investigate microRNA profile of cancer stem-like cells (CSLCs) isolated form freshly resected larynx cancer (LCa) tissue samples.

CD133 positive (CD133⁺) stem-like cells were isolated from freshly resected LCa tumor specimens. MicroRNA profile of 12 pair of CD133⁺ and CD133⁻ cells was determined using microRNA microarray and differential expressions of selvected microRNAs were validated by quantitative real time PCR (qRT-PCR).

MicroRNA profiling of CD133⁺ and CD133⁻ LCa samples with microarray revealed that miR-26b, miR-203, miR-200c, and miR-363-3p were significantly downregulated and miR-1825 was upregulated in CD133⁺ larynx CSLCs. qRT-PCR analysis in a total of 25 CD133⁺/CD133⁻ sample pairs confirmed the altered expressions of these five microRNAs. Expressions of miR-26b, miR-200c, and miR-203 were significantly correlated with miR-363-3p, miR-203, and miR-363-3p expressions, respectively. Furthermore, in silico analysis revealed that these microRNAs target both cancer and stem-cell associated signaling pathways.

Our results showed that certain microRNAs in CD133⁺ cells could be used as cancer stem cell markers. Based on these results, we propose that this panel of microRNAs might carry crucial roles in LCa pathogenesis through regulating stem cell properties of tumor cells.

The online version of this article (doi:10.1186/s12885-016-2863-3) contains supplementary material, which is available to authorized users.

• CD133
• Cancer stem-like cells
• Larynx cancer
• MicroRNAs
• microRNA-signature

Reliable, scalable and time-efficient culture methods are required to fully realize the clinical and industrial applications of human pluripotent stem (hPS) cells. Here we present a completely defined, xeno-free medium that supports long-term propagation of hPS cells on uncoated tissue culture plastic. The medium consists of the Essential 8 (E8) formulation supplemented with inter-α-inhibitor (IαI), a human serum-derived protein, recently demonstrated to activate key pluripotency pathways in mouse PS cells. IαI efficiently induces attachment and long-term growth of both embryonic and induced hPS cell lines when added as a soluble protein to the medium at seeding. IαI supplementation efficiently supports adaptation of feeder-dependent hPS cells to xeno-free conditions, clonal growth as well as single-cell survival in the absence of Rho-associated kinase inhibitor (ROCKi). This time-efficient and simplified culture method paves the way for large-scale, high-throughput hPS cell culture, and will be valuable for both basic research and commercial applications.

Improved culture methods are needed to reliably grow human pluripotent stem cells (hPSCs) on a large scale. Here, the authors identify a xeno-free medium with a supplement of Inter-α-inhibitor that supports long-term propagation and improved single-cell passaging of hPSCs on uncoated plastic.

• Aneuploidy
• Cell therapy
• Embryonic stem cells
• Induced pluripotent stem cells
• Regenerative medicine
• Tissue engineering

• Aneuploidy
• Animals
• Cell Culture Techniques/methods
• DNA Methylation
• Embryonic Stem Cells/cytology
• Embryonic Stem Cells/metabolism
• Epigenesis, Genetic
• Genomic Instability
• Humans
• Microsatellite Instability
• Pluripotent Stem Cells/cytology
• Pluripotent Stem Cells/metabolism
• Tissue Engineering/methods

• Caprine mesenchymal
• Osteogenesis stem cells

Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.

•

Increased culture density induces DNA damage and genomic alterations in hESC

Regenerative medicine is a rapidly growing field that holds promise for the treatment of many currently unresponsive diseases. Stem cells (SCs) are undifferentiated cells with long-term self-renewal potential and the capacity to develop into specialized cells. SC-based therapies constitute a novel and promising concept in regenerative medicine. Radiotherapy is the most frequently used method in the adjuvant treatment of tumorous alterations. In the future, the usage of SCs in regenerative medicine will be affected by their regular and inevitable exposure to ionizing radiation (IR). This phenomenon will be observed during treatment as well as diagnosis. The issue of the genetic stability of SCs and cells differentiated from SCs is crucial in the context of the application of these cells in clinical practice. This review examines current knowledge concerning the DNA repair mechanisms (base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end-joining) of SCs in response to the harmful effects of genotoxic agents such as IR and chemotherapeutics.

• DNA damage response
• DNA repair
• chemotherapeutics
• genetic stability
• ionizing radiation
• pluripotent stem cells