ABSTRACT
The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.
Subject(s)
Stem Cell Research , Humans , Reproducibility of ResultsABSTRACT
The spinal cord emerges from a niche of neuromesodermal progenitors (NMPs) formed and maintained by WNT/fibroblast growth factor (FGF) signals at the posterior end of the embryo. NMPs can be generated from human pluripotent stem cells and hold promise for spinal cord replacement therapies. However, NMPs are transient, which compromises production of the full range of rostrocaudal spinal cord identities in vitro. Here we report the generation of NMP-derived pre-neural progenitors (PNPs) with stem cell-like self-renewal capacity. PNPs maintain pre-spinal cord identity for 7-10 passages, dividing to self-renew and to make neural crest progenitors, while gradually adopting a more posterior identity by activating colinear HOX gene expression. The HOX clock can be halted through GDF11-mediated signal inhibition to produce a PNP and NC population with a thoracic identity that can be maintained for up to 30 passages.
Subject(s)
Neural Crest , Pluripotent Stem Cells , Body Patterning/genetics , Bone Morphogenetic Proteins/metabolism , Cell Differentiation/genetics , Fibroblast Growth Factors/metabolism , Growth Differentiation Factors/metabolism , Humans , Neural Crest/metabolism , Pluripotent Stem Cells/metabolism , Spinal Cord/metabolismABSTRACT
The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the protein antigens CD9, Thy1 (also known as CD90), tissue-nonspecific alkaline phosphatase and class 1 HLA, as well as the strongly developmentally regulated genes NANOG, POU5F1 (formerly known as OCT4), TDGF1, DNMT3B, GABRB3 and GDF3. Nevertheless, the lines were not identical: differences in expression of several lineage markers were evident, and several imprinted genes showed generally similar allele-specific expression patterns, but some gene-dependent variation was observed. Also, some female lines expressed readily detectable levels of XIST whereas others did not. No significant contamination of the lines with mycoplasma, bacteria or cytopathic viruses was detected.
Subject(s)
Embryonic Stem Cells/cytology , Gene Expression Regulation, Developmental , Alkaline Phosphatase/metabolism , Antigens, CD/biosynthesis , Biotechnology/methods , Cell Differentiation , Cell Lineage , Cell Membrane/metabolism , Cells, Cultured , Cluster Analysis , Female , Gene Expression Profiling , Genotype , Glycolipids/chemistry , Humans , Membrane Glycoproteins/biosynthesis , Tetraspanin 29ABSTRACT
The use and banking of biological material for research or clinical application is a well-established practice. The material can be of human or non-human origin. The processes involved in this type of activity, from the sourcing to receipt of materials, require adherence to a set of best practice principles that assure the ethical and legal procurement, traceability, and quality of materials.
Subject(s)
Biological Specimen Banks/legislation & jurisprudence , Tissue Banks/legislation & jurisprudence , Animals , Humans , ResearchABSTRACT
In the 10 years that the technology to produce human embryonic stem cell lines has been available, hundreds of lines have been derived in numerous global locations. These cell lines are being used by researchers across diverse scientific fields to investigate the basic biology, clinical potential, and pharmaceutical applications of these cells and their progeny. In this fast-moving and rapidly growing field, how can we ensure that data generated by different laboratories using the same cell lines are comparable, reproducible, and consistent? One suggestion would be to ensure the quality of the "seed stock" material received and used by researchers. Because a number of laboratories worldwide provide stem cell lines to the scientific community, it seems logical to explore the harmonization of practices between distributors to establish cohesive standards and aid the global movement of stem cell lines to the research community. In the future, when these cells arrive in the clinic for therapeutic use, this consensus of "best practice" should ensure the consistency and facilitate the dissemination of these valuable materials.
Subject(s)
International Cooperation/legislation & jurisprudence , Stem Cells/cytology , Tissue Banks , Embryo Research , Humans , Quality Control , Stem Cells/physiology , Tissue Banks/legislation & jurisprudence , Tissue Banks/organization & administrationABSTRACT
Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins ( approximately 5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase-PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10(-4) to 10(-3)) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.