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PubMed1992

A model of the control of cellular regeneration in the intestinal crypt after perturbation based solely on local stem cell regulation.

The control mechanisms involved in regeneration of murine intestinal crypts after perturbations are presently not well understood. The existence of some feedback signals from the cells on the villus to the cells in the crypt has been suggested. However, some recent experimental data point to the fact that regeneration in the crypt starts very early after perturbation, at a time when the villus cell population has hardly changed. In particular, this early cell proliferative activity is seen specifically at the bottom of the crypt, i.e. in the presumed stem cell zone and furthest from the villus. The objective of this study was to investigate whether a new concept of regulation operating solely at the stem cell level could explain the present mass of accumulated data on the post-irradiation recovery, which is an extensively studied perturbation from the experimental point of view. In order to check its validity, the new concept was formalized as a mathematical simulation model thus enabling comparison with experimental data. The model describes the cellular development from stem cells to the mature villus cells. As a basic feature it is assumed that the self-maintenance and the cell cycle activity of the stem cells are controlled by the number of these cells in an autoregulatory fashion. The essential features of the experimental data (i.e. the recovery with time and the consistency between different types of measurements) can be very well reproduced by simulations using a range of model parameters. Thus, we conclude that stem cell autoregulation is a valid concept which could replace the villus crypt feedback concept in explaining the early changes after irradiation when the damage primarily affects the crypt. The question of the detailed nature of the control process requires further investigation.

Animals↗
PubMed2024

Hepatitis B Virus-KMT2B Integration Drives Hepatic Oncogenic Processes in a Human Gene-edited Induced Pluripotent Stem Cells-derived Model.

BACKGROUND & AIMS: Hepatitis B virus (HBV)-DNA integration into the host genome contributes to hepatocellular carcinoma (HCC) development. KMT2B is the second most frequent locus of HBV-DNA integration in HCC; however, its role and function remain unclear. We aimed to clarify the impact of HBV-KMT2B integration in HCC development using a human genome-edited induced pluripotent stem cell (iPSCs) model. METHODS: Based on the genetic information on HBV-KMT2B integration in HCC, we determined its complete DNA sequence and transcript variants. To exclude the effect of other oncogenic mutations, we reproduced HBV integration in healthy donor iPSCs with an intact genome and analyzed its effects using iPSC-derived hepatic progenitor cells (HPCs) and hepatocytes (iPS-Heps). RESULTS: The reproduced HBV-KMT2B integration significantly upregulated the proliferation of hepatic cells. Comprehensive transcriptional and epigenetic analyses revealed enhanced expression of cell cycle-related genes in hepatic cells with HBV-KMT2B integration based on perturbation of histone 3 lysine 4 tri-methylation (H3K4me3), mimicking that in the original HCC sample. Long-read RNA-sequence detected the common KMT2B transcript variants in the HCC sample and HPCs. Overexpression of the truncated variant significantly enhanced proliferation of hepatic cells, whereas HBV-KMT2B fusion transcripts did not enhance proliferation. HBV-KMT2B-integrated HPCs exhibited replication stress and DNA damage, indicating that our model initiated the process of hepatocarcinogenesis due to abnormally promoted KMT2B function. CONCLUSIONS: Our disease model using genetically engineered iPSCs provides the first insight into both the KMT2B function in HCC development and the oncogenic processes by HBV-KMT2B integration. We clarified the novel oncogenic mechanism in HBV-related HCC due to aberrant KMT2B function.

Humans↗
PubMed1995

Presence of nerve growth factor and its receptors in an in vitro model of islet cell development: implication in normal islet morphogenesis.

In vivo, the differentiation of pancreatic islet stem cells depends on unknown soluble factors produced by the mesenchyme surrounding these cells. We have previously demonstrated that, like some neuronal cells, different beta-cell lines express functional nerve growth factor (NGF) receptors and can respond to NGF by extending neurite-like processes. NGF receptors are also expressed in vivo in mature rat islets and early during development in pancreatic ductular cells, which represent putative beta-stem cells. In this study, we have further characterized an in vitro model of islet development and studied the expression of NGF receptors and its ligand in this model. We have demonstrated the expression of Trk-A messenger RNA coding for the high affinity NGF receptor in islet cells and the localization of Trk protein in both alpha- and beta-islet cells. Moreover, the cells, from which islet cells "bud," also express Trk-A. Furthermore, NGF is produced and secreted by the nonendocrine cells surrounding the islets, suggesting a possible paracrine mode of action of NGF on the adjacent islet cells. Finally, islet morphogenesis is significantly retarded in the presence of K252a, an inhibitor of the tyrosine kinase activity of the family of Trk receptors, suggesting an implication of the neurotrophin-neurotrophin receptor axis in islet development.

Animals↗
PubMed2005

Kinetics and symmetry of divisions of hematopoietic stem cells.

To fulfill the dual abilities to self-renew and to differentiate into cells of multiple lineages, stem cells must undergo, at some stage, asymmetric divisions to generate cells to sustain the stem cell pool as well as the various progeny cells of the distinct lineages. A central question in developmental biology is how a single cell can divide to produce two progeny cells that adopt different fates. Different daughter cells can theoretically arise by uneven distribution of determinants upon cell division, i.e., due to intrinsic factors, or become different upon subsequent exposure to environmental signals, i.e., due to extrinsic factors. Recent advances in the understanding of stem cell biology in Drosophila and murine models have served as a model for hematopoietic stem cell (HSC) development. Provided with advances in molecular and cellular biology, we have gained insight into the mechanisms governing self-renewing asymmetric divisions of primitive HSC. Direct contact with cellular determinants in the niche has been shown to play an essential role in the balance between self-renewing asymmetric division versus differentiation. Identification of the molecular interactions between stem cells and their niche will lead to an understanding of the mechanisms controlling the long-term destiny of stem cells. Ultimately, molecular signals triggered by adhesion and junction complexes are probably responsible for the specific adoption of differentiation pathways.

Animals↗
PubMed2005

Generating chimeric spinal cord: a novel model for transplantable oligodendrocyte progenitors derived from embryonic stem cells.

OBJECT: To identify and evaluate stem cell-derived oligodendrocytes obtained for cell transplantation therapies, the authors developed a novel model to examine single, adult oligodendrocytes in situ. METHODS: Green fluorescent protein-expressing, mouse embryonic stem cells (ESCs) were neural induced and additionally staged in an oligosphere preparatory step for high-yield derivation of oligodendrocyte progenitors. These transplantable, induced progenitors were injected into postnatal Day 2 rat pups, in which spinal cord sections were then examined at 3 and 9 weeks posttransplantation. CONCLUSIONS: Transplanted oligosphere ESCs survived and integrated anatomically into postnatal and adult white matter, generating targeted regions of chimeric spinal cord. A simple model for identifying adult oligodendrocytes in situ is presented, which is suitable for use in further studies examining functional myelination and derivation of oligodendrocytes from genetically engineered ESC lines, including human ESCs. Results from the model presented here demonstrate a unique method for examining transplantable oligodendrocyte progenitors derived from ESCs for repair of white matter disease.

Animals↗
PubMed2005

Evolutionary dynamics of adult stem cells: comparison of random and immortal-strand segregation mechanisms.

This paper develops a point-mutation model describing the evolutionary dynamics of a population of adult stem cells. Such a model may prove useful for quantitative studies of tissue aging and the emergence of cancer. We consider two modes of chromosome segregation: (1) random segregation, where the daughter chromosomes of a given parent chromosome segregate randomly into the stem cell and its differentiating sister cell and (2) "immortal DNA strand" co-segregation, for which the stem cell retains the daughter chromosomes with the oldest parent strands. Immortal strand co-segregation is a mechanism, originally proposed by [Cairns Nature (London) 255, 197 (1975)], by which stem cells preserve the integrity of their genomes. For random segregation, we develop an ordered strand pair formulation of the dynamics, analogous to the ordered strand pair formalism developed for quasispecies dynamics involving semiconservative replication with imperfect lesion repair (in this context, lesion repair is taken to mean repair of postreplication base-pair mismatches). Interestingly, a similar formulation is possible with immortal strand co-segregation, despite the fact that this segregation mechanism is age dependent. From our model we are able to mathematically show that, when lesion repair is imperfect, then immortal strand co-segregation leads to better preservation of the stem cell lineage than random chromosome segregation. Furthermore, our model allows us to estimate the optimal lesion repair efficiency for preserving an adult stem cell population for a given period of time. For human stem cells, we obtain that mispaired bases still present after replication and cell division should be left untouched, to avoid potentially fixing a mutation in both DNA strands.

Adult↗
PubMed2005

Characterization of the interactions between stromal and haematopoietic progenitor cells in expansion cell culture models.

Development of the long-term culture models of haematopoietic stem cells (HSCs) is one of the important tasks in modern biotechnology. It has been suggested that stromal presence is important for haematopoiesis in vitro and in vivo, but the question remains: whether diffusible factors produced by stromal cells are sufficient for the regeneration of primitive and definitive haematopoietic cells, or direct cell-to-cell contacts of the cultured material with underlying stromal base would be required. During present studies, influence of various feeder layers and feeder layer conditioned media on proliferative, differentiative and clonogenic activity of human AC133+ derived from human umbilical cord blood was investigated. Cell extracts for feeder layers were prepared from 4-6 weeks old human embryos and co-cultured feeder cells. Effects of the conditioned media were also determined. Culture and feeder layer media were additionally supplemented with commonly implemented factors such as GM-CSF, IL-3 and LIF. Estimation of morpho-functional properties of AC133+ cultivated suspension cultures was performed in subculture experiments using semisolid agar culture conditions. Multipotential CFU-MIX (CFU-GEMM) and unipotential progenitor cells CFU-GM, BFU-E and CFU-E were observed and analyzed. Our data suggest that haematopoiesis can be sustained for prolonged cultivation periods in the presence of feeder layer cells or conditioned media supported culture models. Prolonged support of primitive haematopoietic cells and their clonogenic capacity and functional characteristics in feeder layer positive cultures, indicates that diffusible factors are sufficient for haematopoiesis and suggests that direct cell-to-cell contacts may not be exclusively required for successful long-term in vitro haematopoiesis.

Cell Communication↗
PubMed2002

Functional human T lymphocyte development from cord blood CD34+ cells in nonobese diabetic/Shi-scid, IL-2 receptor gamma null mice.

An experimental model for human T lymphocyte development from hemopoietic stem cells is necessary to study the complex processes of T cell differentiation in vivo. In this study, we report a newly developed nonobese diabetic (NOD)/Shi-scid, IL-2Rgamma null (NOD/SCID/gamma(c)(null)) mouse model for human T lymphopoiesis. When these mice were transplanted with human cord blood CD34(+) cells, the mice reproductively developed human T cells in their thymus and migrated into peripheral lymphoid organs. Furthermore, these T cells bear polyclonal TCR-alphabeta, and respond not only to mitogenic stimuli, such as PHA and IL-2, but to allogenic human cells. These results indicate that functional human T lymphocytes can be reconstituted from CD34(+) cells in NOD/SCID/gamma(c)(null) mice. This newly developed mouse model is expected to become a useful tool for the analysis of human T lymphopoiesis and immune response, and an animal model for studying T lymphotropic viral infections, such as HIV.

Animals↗
PubMed2003

Patterns of cell division and the risk of cancer.

Epidermal and intestinal tissues divide throughout life to replace lost surface cells. These renewing tissues have long-lived basal stem cell lineages that divide many times, each division producing one stem cell and one transit cell. The transit cell divides a limited number of times, producing cells that move up from the basal layer and eventually slough off from the surface. If mutation rates are the same in stem and transit divisions, we show that minimal cancer risk is obtained by using the fewest possible stem divisions subject to the constraints imposed by the need to renew the tissue. In this case, stem cells are a necessary risk imposed by the constraints of tissue architecture. Cairns suggested that stem cells may have lower mutation rates than transit cells do. We develop a mathematical model to study the consequences of different stem and transit mutation rates. Our model shows that stem cell mutation rates two or three orders of magnitude less than transit mutation rates may favor relatively more stem divisions and fewer transit divisions, perhaps explaining how renewing tissues allocate cell divisions between long stem and short transit lineages.

Cell Division↗
PubMed2003

Modeling stem cell population growth: incorporating terms for proliferative heterogeneity.

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle-derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Apoptosis↗
PubMed2004

Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro.

A major area in regenerative medicine is the application of stem cells in bone reconstruction and bone tissue engineering. This will require well-defined and efficient protocols for directing the differentiation of stem cells into the osteogenic lineage, followed by their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages on transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying osteogenesis and bone development, and facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmokinetic and cytotoxicity/genotoxicity screening tests for bone-related biomaterials and drugs could also use protocols developed for the osteogenic differentiation of stem cells. This review critically examines the various strategies that could be used to direct the differentiation of stem cells into the osteogenic lineage in vitro.

Animals↗
PubMed2006

Regulation of human neural precursor cells by laminin and integrins.

Deciphering the factors that regulate human neural stem cells will greatly aid in their use as models of development and as therapeutic agents. The extracellular matrix (ECM) is a component of stem cell niches in vivo and regulates multiple functions in diverse cell types, yet little is known about its effects on human neural stem/precursor cells (NSPCs). We therefore plated human NSPCs on four different substrates (poly-L-ornithine, fibronectin, laminin, and matrigel) and compared their responses with those of mouse NSPCs. Compared with the other substrates, laminin matrices enhanced NSPC migration, expansion, differentiation into neurons and astrocytes, and elongation of neurites from NSPC-derived neurons. Laminin had a similar spectrum of effects on both human and mouse cells, highlighting the evolutionary conservation of NSPC regulation by this component of the ECM. Flow cytometry revealed that human NSPCs express on their cell surfaces the laminin-binding integrins alpha3, alpha6, alpha7, beta1, and beta4, and function-blocking antibodies to the alpha6 subunit confirmed a role for integrins in laminin-dependent migration of human NSPCs. These results define laminin and its integrin receptors as key regulators of human NSPCs.

Animals↗
PubMed1997

Immune-deficient SCID and NOD/SCID mice models as functional assays for studying normal and malignant human hematopoiesis.

Many events and requirements of the developmental program of human hematopoietic stem cells have not yet been discovered. A major impediment has been the lack of an appropriate experimental system. At present the conditions for maintaining human stem cells in vitro are not fully known. As a result within a short period the small stem cell pool is lost due to differentiation, making it difficult to examine the correlation between these cells and their function in vivo. Most of our knowledge of hematopoietic stem cells is from animal models in which purified stem cell canididates are assayed based on their functional ability to rescue lethally conditioned recipients. The permanent correction of many genetic disorders of the hematopoietic system requires efficient methods for introducing genes into stem cells in vitro. However, progress has been hindered by the absence of preclinical models that assay the repopulating capacity of primitive human cells. In addition, the development of therapy for malignant diseases also requires assays to identify the target leukemic stem cells based on their ability to initiate the disease. The recent development of methods to transplant or implant both normal and leukemic cells into immune-deficient mice provides the foundation for human stem cell assays. These models assay the repopulating capacity of primitive human cells and provide an important approach to identify and characterize human stem cells, both normal and leukemic. This review focuses on the development of functional assays for normal and leukemic human stem cells and on the new insights that these models are beginning to provide on the organization of the human stem cell hierarchy.

Animals↗
PubMedDate not supplied

Transduction of a preselected population of human epidermal stem cells: consequences for gene therapy.

Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.

Adult↗
PubMed1993

Embryonic stem cell model systems for vascular morphogenesis and cardiac disorders.

To better understand the formation of the cardiovascular system and its disease states, models amenable to manipulation must be developed. In this article we present two models. One is a small animal model for an inflammatory disorder that can lead to heart failure. Production of this model is based on the ability of blastocyst-derived embryonic stem cells, which can be genetically altered in vitro by a technique called gene targeting, to reconstitute an entire animal when reintroduced into a blastocyst and allowed to colonize the germ line of the resulting chimeric embryo. The other model is based on the capacity of embryonic stem cells to differentiate in culture into embryo-like structures called embryoid bodies. Embryoid bodies contain angioblasts, or prevascular endothelial cells, which can be induced to undergo aspects of vascular development by manipulation of culture conditions.

Animals↗
PubMed2004

Use and application of stem cells in toxicology.

In recent years, stem cells have been the subject of increasing scientific interest because of their utility in numerous biomedical applications. Stem cells are capable of renewing themselves; that is, they can be continuously cultured in an undifferentiated state, giving rise to more specialized cells of the human body such as heart, liver, bone marrow, blood vessel, pancreatic islet, and nerve cells. Therefore, stem cells are an important new tool for developing unique, in vitro model systems to test drugs and chemicals and a potential to predict or anticipate toxicity in humans. The following review provides an overview of the applications of stem cell technology in the area of toxicology. Specifically, this review addresses core technologies that are emerging in the field and how they could fulfill critical safety issues such as QT prolongation and hepatotoxicity, two leading causes of failures in preclinical development of new therapeutic drugs. We report how adult stem cells derived from various sources, such as human bone marrow and placenta, can potentially generate suitable models for cardiotoxicity, hepatotoxicity, genotoxicity/epigenetic and reproductive toxicology screens. Additionally, this review addresses the role and advantages of embryonic stem cells in the aforementioned models for toxicity and how genetic selection is employed to overcome major limitations to the implementation of stem cell-based in vitro models for toxicology.

Adult↗