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Plasticity of Adult Bone Marrow Stem Cells
Abstract
In mammals, including humans, newly differentiated cells are continuously generated from stem cells throughout development. In the adult, stem cells are found in different organ systems where they can contribute to the replacement of cells lost to physiological turnover, injury, or disease. When taken from their residence bone marrow-derived stem cells develop characteristics that typify brain, muscle, liver, and endothelial cells. Analogously, brain-derived stem cells exhibit characteristics of hematopoietic and muscle cells. The fate of a cell is therefore likely to be dictated in part by the local environment. This chapter reviews the bone marrow stem cell plasticity, focusing on the differentiation of the cells into different neural cell types and their potential therapeutic value in the treatment of central nervous system (CNS) injury and disease. Stem cells change their properties over time. In the immune system, fetal hematopoietic stem cells have different antigenic properties and exhibit broader lineage potentials than do adult hematopoietic stem cells.
... Two characteristics distinguish them from other cell types: first, they are the never ending source of parental cells needed for continuous replication of specialized cells, i.e. cells terminally differentiated into many cell populations (Stem cells, 2001). A good example of this is the gut epithelial cell; the rapid rate of cell turnover throughout the intestinal tract demands that undif-ferentiated stem cells constantly produce new epithelial cells (Chandross and Mezey, 2001). Second, stem cells have to give rise to different cell types in response to local signals (Stem cells, 2001;Slack, 2000). ...
... myocytes in muscle, or insulin-producing cells of the pancreas) (Stem cells, 2001). Stem cells are therefore required to have the potential to develop into many different cell types in the body, and the potential to replace injured tissues (Stem cells, 2001;Chandross and Mezey, 2001;Slack, 2000;Zhao et al., 2003). When a stem cell divides, a new cell has the potential either to remain a stem cell or to become another type of cell with a more specialized function, such as a muscle cell, a red blood cell or a neuron (Stem cells, 2001;Uzan, 2004). ...
... From this bulge all tissues originate, i.e. it allows for the differentiation of multiple specialized cell types that will generate the heart, lung, skin and all other tissues (Stem cells, 2001;Buttery et al., 2001;Brook and Gardner, 1997;Bongso et al., 1994a;Bongso et al., 1994b;Bongso et al., 1995;Bongso, 1996;Itskovitz-Eldor et al., 2000;Thomson and Odorico, 2000;Pedersen, 1999;Brustle et al., 1999;Thomson et al., 1998;Reubinoff et al., 2000;Odorico et al., 2001;Doetschman et al., 1985;Marshak et al., 2001;Call et al., 2000). In addition, in certain adult tissues, such as in bone marrow, liver, muscle and brain, discrete existing populations of adult pluripotent stem cells generate replacements for individual cells lost through normal aging processes, injury, or diseases (Stem cells, 2001;Chandross and Mezey, 2001;Slack, 2000;Zhao et al., 2003;Uzan, 2004;Draper et al., 2004). It has been proposed that stem cells will become the basis for treating chronic and degenerative diseases. ...
Even in the absence of damage or illness mature animals need billions of new cells every single day of their lives in order to survive and renew circulating blood cells and intestinal and skin lining. This task is accomplished by undifferentiated cells residing in most adult organs. These cells are designated adult stem cells (ASC) since they represent the adult counterpart, present in almost every organ, of the embryonal stem cells (ES) from which the entire human body develops. Scientists first hypothesized the existence of stem cells over a century ago, and haematopoietic stem cells (HSC) have been exploited for the therapy of human diseases for two decades. Other types of stem cells also circulating in the bloodstream have been described. We briefly describe the potential uses of each of these types of cells, including autologous circulating stem cells, for disease therapy and in particular for the possible reversal of liver failure due to chronic hepatitis and/or cirrhosis.
... Events connect the outer world and the inner feeling affect tissues. Cells derived from the mesoderm, which lies between the endoderm and the ectoderm, give rise to all other tissues of the body, including the dermis of the skin, adrenal cortex, lymphatic tissue, skeletal, smooth and cardiac muscle, connective tissues (e.g., bone, cartilage), urogenital system, heart, and blood vessels [20]. Most organs have three representative germ layers. ...
... Fundamental cells are capable of producing on one another, with the singular purpose of recreating itself for homeostasis. This is only a basic element of the cell, but in most cases, especially the subject here within, ESCs are capable of several layers of differentiation [134]. Unique from other cell lines, ESCs have been specially developed to recreate several tissue layers in order to animate an organism inside of the fetus. ...
Parkinson’s disease, type 1 diabetes, and coronary artery disease are some of the few difficult diseases to control. As a result, there has been pressure in the scientific community to develop new technologies and techniques that can treat, or ultimately cure these life-threatening diseases. One such scientific advancement in bridging the gap is the use of stem cell therapy. In recent years, stem cell therapy has gained the spotlight in becoming a possible intervention for combating chronic diseases due to their unique ability to differentiate into almost any cell line. More precisely, embryonic stem cell therapy may hold the potential for becoming the ideal treatment for a multitude of diseases as embryonic stem cells are not limited in their ability to differentiate like their counterpart adult stem cells. Although there has been controversy around the usage of embryonic stem cells, there has been found a great deal of potential within the usage of these cells to treat a multitude of life-threatening diseases. In this article, we will break down the categories of diseases in which embryonic stem cell therapy can be applied into: autoimmune, neurological, and cardiovascular with three diseases relating to each category. Our aim is to provide a comprehensive review on the advantages of embryonic stem cells (ESCs) that can solve current obstacles and push advances towards stem cell therapies in the field for the most common diseases.
... Stem cells play many vital roles in our body, they are long lived, uncommon, mother cells which have ability to self renew. [3] Self renewal, one of the defining characteristics of the stem cells, is a cell division in which one or both of the resulting daughter cells remains undifferentiated, retaining the ability to give rise to another stem cell with the same capacity to proliferate as the parental cell [4] and have the potential to become any type of cell, they can become cells of the blood, heart, bones, skin, muscles, brain etc. [5]. When mutated, they can potentially become cancer stem cells (CSCs). ...
... Other cells, which are essential for embryonic development but are not incorporated into the body of the embryo, include the extra embryonic tissues, placenta, and umbilical cord. 1 Most investigators use the term pluripotent (derived from the Latin plures ¼ many) to describe stem cells that can give rise to cells derived from all three embryonic germ layersmesoderm, endoderm, and ectoderm. 13 The germ layers are the embryonic source of all cells of the human body. All of the different types of specialized cells are derived from one of these germ layers, a property observed in the natural course of embryonic development under certain laboratory conditions. ...
... Other cells, which are essential for embryonic development but are not incorporated into the body of the embryo, include the extra embryonic tissues, placenta, and umbilical cord. 1 Most investigators use the term pluripotent (derived from the Latin plures ¼ many) to describe stem cells that can give rise to cells derived from all three embryonic germ layersmesoderm, endoderm, and ectoderm. 13 The germ layers are the embryonic source of all cells of the human body. All of the different types of specialized cells are derived from one of these germ layers, a property observed in the natural course of embryonic development under certain laboratory conditions. ...
... The embryonic stem cell is pluripotent-it can give rise to cells derived from all three germ layers. Multipotent cell or precursor cell, such as the hematopoietic cell, is one with a more limited range of differentiation and can give rise to cell types whose lineage can be traced back to only one primary germ layer (Figures 1 and 2) (Chandross and Mezey, 2001). ...
Stem cell technology has developed rapidly in recent years to the point that we can now envisage its future use in a variety of therapeutic areas. This review seeks to summarize the types and sources of stem cells that may be utilized in this way, their pattern of development, their plasticity in terms of differentiation and transdifferentiation, their ability to self-renew, the privileged microenvironment in which they are housed, their cell surface markers used to track them, issues relating to their transfection, and their fate. Particular reference is made, as prime examples, to how both the function of mesenchymal and neural stem cells are being studied experimentally, and currently used clinically in certain circumstances, towards the ultimate aim of their mainstream therapeutic use.
... Other cells, which are essential for embryonic development but are not incorporated into the body of the embryo, include the extra embryonic tissues, placenta, and umbilical cord. 1 Most investigators use the term pluripotent (derived from the Latin plures ¼ many) to describe stem cells that can give rise to cells derived from all three embryonic germ layersmesoderm, endoderm, and ectoderm. 13 The germ layers are the embryonic source of all cells of the human body. All of the different types of specialized cells are derived from one of these germ layers, a property observed in the natural course of embryonic development under certain laboratory conditions. ...
Different types of stem cells have a role in liver regeneration or fibrous repair during and after several liver diseases. Otherwise, the origin of hepatic and/or extra-hepatic stem cells in reactive liver repopulation is under controversy. The ability of the human body to self-repair and replace the cells and tissues of some organs is often evident. It has been estimated that complete renewal of liver tissue takes place in about a year. Replacement of lost liver tissues is accomplished by proliferation of mature hepatocytes, hepatic oval stem cells differentiation, and sinusoidal cells as support. Hepatic oval cells display a distinct phenotype and have been shown to be a bipotential progenitor of two types of epithelial cells found in the liver, hepatocytes, and bile ductular cells. In gastroenterology and hepatology, the first attempts to translate stem cell basic research into novel therapeutic strategies have been made for the treatment of several disorders, such as inflammatory bowel diseases, diabetes mellitus, celiachy, and acute or chronic hepatopaties. In the future, pluripotent plasticity of stem cells will open a variety of clinical application strategies for the treatment of tissue injuries, degenerated organs. The promise of liver stem cells lie in their potential to provide a continuous and readily available source of liver cells that can be used for gene therapy, cell transplant, bio-artificial liver-assisted devices, drug toxicology testing, and use as an in vitro model to understand the developmental biology of the liver.
... Termin ten oznacza, ˝e komórka macierzysta jednej tkanki mo˝e ró˝nicowaç si´ w dojrza∏à komórkí nnej tkanki in vitro [42,50]. Potwierdzajàc ten fakt, udowodniono, ˝e komórki macierzyste krwi pochodzenia mezodermalnego mogà utworzyç miocyty, tak˝e mezodermalne, a tak˝e wywodzàce si´ z ektodermy, neurony [1, 16,25,50]. Natomiast komórki macierzyste uk∏adu nerwowego mogà si´ ró˝nicowaç w komórki krwi [11]. Przeprowadzone pod tym kàtem badania obali∏y tez´, i˝ tylko embrionalne komórki macierzyste sà zdolne do ró˝nicowania si´ w komórki wi´cej ni˝ jednej tkanki. ...
Enormous hope is connected with stem cells with regard to cell therapy, and this has become one of the most dynamically developing areas of science at the moment. A stem cell has unlimited potential for self-renewal. It appears that it can be a source of in vitro differentiated progeny cells capable of repairing damaged tissue. These review provides information about the biological properties of embryonic stem cells, i.e. ESs (embryonic stem cells), EGs (embryonic germ cells), and ECs (embryonic carcinoma cells). Possible human embryonic stem cell applications are described, with consideration of the desired cell line and the signals involved in their differentiation. The information about adult stem cells present - hemopoietic stem cells and the cells residing in selected tissues and organs: endothelium, pancreas, liver, epithelium, and gastrointestinal tract. Methods of their identification using the cell surfaces are also presented: the possibilities of in vitro transdifferentation, the phenomenon of in vivo plasticity, as well as morphological and genetic properties. Some topics of cell therapy and its clinical application in diabetics amplification are included.
The goal in transplant therapy is to provide adequate numbers of cells to appropriate sites for useful cellular replacement. In principle, three types of therapy can be considered. One straightforward therapeutic approach is to use dissociated cells for replacement. In this approach, either purified populations of cells or mixed populations of cells can be used. A second therapeutic approach is to develop devices that are a combination of synthetic material and cells. A third possibility is to use stem cells and precursor cells to generate organs in culture. These synthetic organs can then be transplanted. Each of these approaches has been tried with varied amounts of success. Generating organs has been possible for some structures such as skin, blood vessels, lens, and bone (see ref. 1 and references therein), but the inherent complexity of even the simplest neural structures currently renders this an impractical approach for the nervous system. A combination of synthetic material and cells have been used in retinal implants, cochlear replacements, nerve electrode junctions, synthetic neural networks or guidance channels for nerve regrowth. These devices are still in developmental stages and a detailed discussion of such devices is beyond the scope of this chapter. In this chapter, we have limited our discussion to the relative advantages and disadvantages of the various human cell types that are available for dissociated cell replacement therapy.
Looking for the exact definition of stem cell is sometimes the source of endless semantical debates. There are two generally accepted criteria: a high capacity for self-renewal and the potential for multipotent or pluripotent differentiation. Stem cells are unspecialized cells that renew themselves through series of cell divisions, which result in similar unspecialized stem cells. Under certain physiologic or experimental conditions, they can be induced to become differentiated cells with special functions. The resulting progenitor cells differentiate into functional, specialised cells of the organism. In this chapter we provide standard criteria and definitions for stem cells.
Recent advances in regenerative medicine and stem cell therapy has opened new horizons with promising
results in treating diseases so far known as incurable. Therapy with stem cells has been considered in treating
various disorders encompassing hematologic, cardiovascular, neurologic, dermatologic and orthopedic entities
as well as diabetes. Stem cells transplantation has been lately evaluated in treating hearing impairments in
experimental studies and it is expected to open its way in treating patients with sensori-neural hearing loss
in future. The present study is a review of literature published in medical databases current as of July 2007.
Authors in this article has highlighted important characteristics of this novel approach including basics of
stem cell biology and its potential application in treating auditory disorders. Despite primary hopeful results
from stem cells transplantation in treating hearing impairments in experimental research, there are many
questions which should be answered before their being introduced in clinical trials. Finding a proper source
for cellular isolation, method of differentiation, way of delivery to the target organ and the right dosing as well
as the ethical issues and potential hazards confronted are such challenges which should be first overcome.
Biological science is being bombarded with problems of HIV, cancers, thousands of genetic disorders, obesity, diabetes, microbial infections, biological warfare, SARS and degenerative organ diseases of the lung, liver, kidney and heart. A big question today is the promise of stem cells. Will stem cells one day be good enough to save the sinking Noah's Ark of human health? This review attempts to give an overview of stem cells and the scientific factors revolving around it. Keywords: adult stem cells (ASCs), embryonic stem cells (ESCs), human embryonic stem cells (hESCs), hemopoietic stem cells (HESCs), stem cell markers, pluripotency, plasticity IPC Code: Int.Cl. 7 : C12N5/22 Introduction In the 1900s, biological sciences took a big leap in the field of molecular biology, with the discovery of hereditary material DNA, the blue print of life. With the most prestigious project of biology completed today i.e., the 'Human Genome Project', the field of therapy still has numerous unsolved mysteries ranging from treatment of common cold to HIV. In this situation today, stem cells, our own cells from human body give us a chance of hope for a healthier future. The issue of stem cell research burst on the scientific scene in 1998 when researchers reported the isolation of embryonic stem and embryonic germ cells, which offer great promise for new ways of treating diseases. Stem cells are unspecialized or undifferentiated cells that have the unique ability to give rise to many different cell types such as skin, liver, kidney, heart, neuron or other organ cells 1,2 . They also possess the property of self-duplicating for indefinite periods of times and are thus present as adult stem cells in most of the organs of the body including blood and bone marrow 2 . Stem cells are of two types, the adult stem cells as mentioned above and embryonic stem cells, which originate from one of the earliest stages of development of the embryo, the blastocyst stage 3 . More specifically, the embryonic stem cells (ESC's) are derived from the inner cell mass of the blastocyst stage (Fig. 1, Table 1) i.e., before the blastocyst implants itself in the uterine wall. Most importantly, the ESC's are pluripotent. This means that they are capable of self-renewal and differentiating into almost any cell type in the body including the cells of all the three germ layers 3-6 . In contrast, the adult stem cells (ASC's) are unspecialized or undifferentiated cells that are found in differentiated tissues 3 . The ASC's possess the ability to differentiate into the cell type from the tissue of its origin and play a role of replacing damaged cells of the particular tissue or organ. ASC's can be found in the bone marrow, blood stream, cornea and retina of the eye, dental pulp of the tooth, liver, skin, gastrointestinal tract and the pancreas. However, there is not much evidence that ASC's unlike the ESC's are pluripotent 4 . Adult stem cells usually divide to generate progenitor or precursor cells which then differentiate to develop into mature cell types that have characteristic shapes and specialized functions 3,4 . Embryonic stem cells are derived from embryos created through in vitro fertilization (IVF) 17,22 . IVF technology has made it possible to carry out fertilization in vitro and grow embryos in the laboratory 18 . Traditionally, this technology has allowed for many otherwise infertile couples to have children 15 . During this process, the embryos created offer a potential source for human embryonic stem cells 19,20 . However, their use has raised ethical and policy issues, due to which their utilization is restricted.
Even in the absence of damage or illness mature animals need billions of new cells every single day of their lives in order to survive and renew circulating blood cells and intestinal and skin lining. This task is accomplished by undifferentiated cells residing in most adult organs. These cells are designated adult stem cells (ASC) since they represent the adult counterpart, present in almost every organ, of the embryonal stem cells (ES) from which the entire human body develops. Scientists first hypothesized the existence of stem cells over a century ago, and haematopoietic stem cells (HSC) have been exploited for the therapy of human diseases for two decades. Other types of stem cells also circulating in the bloodstream have been described. We briefly describe the potential uses of each of these types of cells, including autologous circulating stem cells, for disease therapy and in particular for the possible reversal of liver failure due to chronic hepatitis and/or cirrhosis.
Mice homozygous for the c14CoS albino deletion die as neonates as a result of liver dysfunction. Previous mapping studies have associated this defect with a 310-kb fragment encoding the hepatocyte-specific developmental regulation locus (alf/hsdr-1). The gene encoding fumarylacetoacetate hydrolase (Fah), a metabolic enzyme that catalyzes the last step of tyrosine catabolism, also maps to the same deletion interval. To test whether the neonatal defects found in the albino deletion mutants are attributable to loss of Fah, and not to another gene mapping to the deletion, we have generated Fah mutant mice by gene targeting in embryonic stem cells. Fah-deficient mice die within 12 hr after birth from hypoglycemia and liver dysfunction. In addition, the same pattern of altered liver mRNA expression found in the albino deletion mutants was also found in affected animals . We conclude that the neonatal lethal and liver dysfunction phenotype of the alf/hsdr-1 deletion is entirely attributable to loss of Fah.
Sustained blood cell production requires preservation of a quiescent, multipotential stem cell pool that intermittently gives rise to progenitors with robust proliferative potential. The ability of cells to shift from a highly constrained to a vigorously active proliferative state is critical for maintaining stem cells while providing the responsiveness necessary for host defense. The cyclin-dependent kinase inhibitor (CDKI), p21cip1/waf1 (p21) dominates stem cell kinetics. Here we report that another CDKI, p27kip1 (p27), does not affect stem cell number, cell cycling, or self-renewal, but markedly alters progenitor proliferation and pool size. Therefore, distinct CDKIs govern the highly divergent stem and progenitor cell populations. When competitively transplanted, p27-deficient stem cells generate progenitors that eventually dominate blood cell production. Modulating p27 expression in a small number of stem cells may translate into effects on the majority of mature cells, thereby providing a strategy for potentiating the impact of transduced cells in stem cell gene therapy.
Mesenchymal stem cells are multipotent cells that can be isolated from adult bone marrow and can be induced in vitro and in vivo to differentiate into a variety of mesenchymal tissues, including bone, cartilage, tendon, fat, bone marrow stroma, and muscle. Despite their potential clinical utility for cellular and gene therapy, the fate of mesenchymal stem cells after systemic administration is mostly unknown. To address this, we transplanted a well-characterized human mesenchymal stem cell population into fetal sheep early in gestation, before and after the expected development of immunologic competence. In this xenogeneic system, human mesenchymal stem cells engrafted and persisted in multiple tissues for as long as 13 months after transplantation. Transplanted human cells underwent site-specific differentiation into chondrocytes, adipocytes, myocytes and cardiomyocytes, bone marrow stromal cells and thymic stroma. Unexpectedly, there was long-term engraftment even when cells were transplanted after the expected development of immunocompetence. Thus, mesenchymal stem cells maintain their multipotential capacity after transplantation, and seem to have unique immunologic characteristics that allow persistence in a xenogeneic environment. Our data support the possibility of the transplantability of mesenchymal stem cells and their potential utility in tissue engineering, and cellular and gene therapy applications.
Remyelination of focal areas of the central nervous system (CNS) in animals can be achieved by transplantation of glial cells, yet the source of these cells in humans to similarly treat myelin disorders is limited at present to fetal tissue. Multipotent precursor cells are present in the CNS of adult as well as embryonic and neonatal animals and can differentiate into lineage-restricted progenitors such as oligodendroglial progenitors (OPs). The OPs present in adults have a different phenotype from those seen in earlier life, and their potential role in CNS repair remains unknown. To gain insights into the potential to manipulate the myelinating capacity of these precursor and/or progenitor cells, we generated a homogenous culture of OPs from neural precursor cells isolated from adult rat subependymal tissues. Phenotypic characterization indicated that these OPs resembled neonatal rather than adult OPs and produced robust myelin after transplantation. The ability to generate such cells from the adult brain therefore opens an avenue to explore the potential of these cells for repairing myelin disorders in adulthood.
The fms-like tyrosine kinase (Flt) is a transmembrane receptor in the tyrosine kinase family. Expression of flt complementary
DNA in COS cells conferred specific, high-affinity binding of vascular endothelial growth factor, also known as vascular permeability
factor (VEGF-VPF), a factor that induces vascular permeability when injected in the guinea pig skin and stimulates endothelial
cell proliferation. Expression of Flt in Xenopus laevis oocytes caused the oocytes to release calcium in response to VEGF-VPF.
These findings show that flt encodes a receptor for VEGF-VPF.
Neurogenesis in the mammalian central nervous system is believed to end in the period just after birth; in the mouse striatum
no new neurons are produced after the first few days after birth. In this study, cells isolated from the striatum of the adult
mouse brain were induced to proliferate in vitro by epidermal growth factor. The proliferating cells initially expressed nestin,
an intermediate filament found in neuroepithelial stem cells, and subsequently developed the morphology and antigenic properties
of neurons and astrocytes. Newly generated cells with neuronal morphology were immunoreactive for gamma-aminobutyric acid
and substance P, two neurotransmitters of the adult striatum in vivo. Thus, cells of the adult mouse striatum have the capacity
to divide and differentiate into neurons and astrocytes.
Cell-transfer studies presented here distinguish three murine B cell lineages: conventional B cells, which develop late and are continually replenished from progenitors in adult bone marrow; Ly-1 B cells (B-1a), which develop early and maintain their numbers by self-replenishment; and Ly-1B "sister" (B-1b) cells, which share many of the properties of Ly-1 B cells, including self-replenishment and feedback regulation of development but can also readily develop from progenitors in adult bone marrow. The sequential emergence of these lineages, the time at which their progenitors function during ontogeny, and the distinctions among their repertoires and functions suggest that evolution has created a layered immune system in which the immune response potential of each successive lineage is adapted to its particular niche.
We have used liver fatty acid-binding protein/human growth hormone (L-FABP/hGH) fusion genes to explore the temporal and spatial differentiation of intestinal epithelial cells in 1- to 12-month-old transgenic mice. The intact, endogenous L-FABP gene (Fabpl) was not expressed in the colon at any time. Young adult transgenic mice containing nucleotides -596 to +21 of the rat L-FABP gene linked to the hGH gene (minus its 5' nontranscribed domain) demonstrated inappropriate expression of hGH in enterocytes and many enteroendocrine cells of most proximal and mid-colonic crypts (glands). Rare patches of hGH-negative crypts were present. With increasing age, a wave of "extinction" of L-FABP (-596 to +21)/hGH expression occurred, first in the distal colon and then in successively more proximal regions, leaving by 10 months of age only rare hGH-positive multicrypt patches. At no time during this progressive silencing of transgene expression were crypts observed that contained a mixture of hGH-positive and -negative cells at a particular cell stratum. Young (5-7 weeks) mice containing a L-FABP (-4000 to +21)/hGH transgene also demonstrated inappropriate expression of the transgene in most proximal colonic crypts. However, the additional 3.3 kilobases of upstream sequence resulted in much more rapid extinction of reporter expression, leaving by 5 months of age only scattered single crypts with detectable levels of hGH. This age-related extinction of L-FABP/hGH expression did not involve enterocytes and enteroendocrine cells in the (proximal) small intestine. These results indicate that cis-acting elements outside of nucleotides -4000 to +21 are necessary to fully modulate suppression of colonic L-FABP expression. They also define fundamental changes in colonic epithelial cell populations during adult life. Our data suggest that (i) a single stem cell gives rise to all cells that populate a given colonic crypt, (ii) stem cells represented in several adjacent crypts may be derived from a common progenitor, and (iii) such a progenitor cell may repopulate colonic crypts with stem cells during adult life. Since each colonic crypt contains the amplified descendants of its stem cell, transgenes may be powerful tools for characterizing the spatial and biological features of gut stem cells and their progenitors during life.
Cells from transgenic mice expressing a human mini-gene for collagen I were used as markers to follow the fate of mesenchymal precursor cells from marrow that were partially enriched by adherence to plastic, expanded in culture, and then injected into irradiated mice. Sensitive PCR assays for the marker collagen I gene indicated that few of the donor cells were present in the recipient mice after 1 week, but 1-5 months later, the donor cells accounted for 1.5-12% of the cells in bone, cartilage, and lung in addition to marrow and spleen. A PCR in situ assay on lung indicated that the donor cells diffusely populated the parenchyma, and reverse transcription-PCR assays indicated that the marker collagen I gene was expressed in a tissue-specific manner. The results, therefore, demonstrated that mesenchymal precursor cells from marrow that are expanded in culture can serve as long-lasting precursors for mesenchymal cells in bone, cartilage, and lung. They suggest that cells may be particularly attractive targets for gene therapy ex vivo.
During the development of the mammalian brain, neuronal precursors migrate to their final destination from their site of birth
in the ventricular and subventricular zones (VZ and SVZ, respectively). SVZ cells in the walls of the lateral ventricle continue
to proliferate in the brain of adult mice and can generate neurons in vitro, but their fate in vivo is unknown. Here SVZ cells
from adult mice that carry a neuronal-specific transgene were grafted into the brain of adult recipients. In addition, the
fate of endogenous SVZ cells was examined by microinjection of tritiated thymidine or a vital dye that labeled a discrete
population of SVZ cells. Grafted and endogenous SVZ cells in the lateral ventricle of adult mice migrate long distances and
differentiate into neurons in the olfactory bulb.
Mice homozygous for the c14CoS albino deletion die as neonates as a result of liver dysfunction. Previous mapping studies have associated this defect with a 310-kb fragment encoding the hepatocyte-specific developmental regulation locus (alf/hsdr-1). The gene encoding fumarylacetoacetate hydrolase (Fah), a metabolic enzyme that catalyzes the last step of tyrosine catabolism, also maps to the same deletion interval. To test whether the neonatal defects found in the albino deletion mutants are attributable to loss of Fah, and not to another gene mapping to the deletion, we have generated Fah mutant mice by gene targeting in embryonic stem cells. Fah-deficient mice die within 12 hr after birth from hypoglycemia and liver dysfunction. In addition, the same pattern of altered liver mRNA expression found in the albino deletion mutants was also found in affected animals. We conclude that the neonatal lethal and liver dysfunction phenotype of the alf/hsdr-1 deletion is entirely attributable to loss of Fah.
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(−8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(−8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
It has been proposed that hematopoietic and endothelial cells are derived from a common cell, the hemangioblast. In this study, we demonstrate that a subset of CD34+ cells have the capacity to differentiate into endothelial cells in vitro in the presence of basic fibroblast growth factor, insulin-like growth factor-1, and vascular endothelial growth factor. These differentiated endothelial cells are CD34+, stain for von Willebrand factor (vWF), and incorporate acetylated low-density lipoprotein (LDL). This suggests the possible existence of a bone marrow-derived precursor endothelial cell. To demonstrate this phenomenon in vivo, we used a canine bone marrow transplantation model, in which the marrow cells from the donor and recipient are genetically distinct. Between 6 to 8 months after transplantation, a Dacron graft, made impervious to prevent capillary ingrowth from the surrounding perigraft tissue, was implanted in the descending thoracic aorta. After 12 weeks, the graft was retrieved, and cells with endothelial morphology were identified by silver nitrate staining. Using the di(CA)n and tetranucleotide (GAAA)n repeat polymorphisms to distinguish between the donor and recipient DNA, we observed that only donor alleles were detected in DNA from positively stained cells on the impervious Dacron graft. These results strongly suggest that a subset of CD34+ cells localized in the bone marrow can be mobilized to the peripheral circulation and can colonize endothelial flow surfaces of vascular prostheses.
All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.
Localization of β-2 integrins in normal and Alzheimer disease temporal cortex was studied immunohistochemically. Resting microglia were found to express constitutively CD11a (LFA-1), CD11b (Mac-1, CR3), CD11c (P150, 95; CR4), and CD18 (β-2). They were also found to express constitutively leukocyte common antigen and the immunoglobulin receptor FcγRI. The intensity of expression of each of these antigens was enhanced on reactive micgrolia in Alzheimer disease tissue. HLA-DR was detected on only a few microglia in control tissue, but was intensely expressed on large numbers of reactive microglia in Alzheimer tissue. These data are consistent with a leukocyte origin and a phagocytic role for microglia. They provide further evidence of an inflammatory response of brain tissue in Alzheimer disease. The microglia were found to make up 9–12% of the total glial population in gray matter and 7.5–9% in white matter.
The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.
A battery of monoclonal antibodies (mAbs) against brain cell nuclei has been generated by repeated immunizations. One of these, mAb A60, recognizes a vertebrate nervous system- and neuron-specific nuclear protein that we have named NeuN (Neuronal Nuclei). The expression of NeuN is observed in most neuronal cell types throughout the nervous system of adult mice. However, some major cell types appear devoid of immunoreactivity including cerebellar Purkinje cells, olfactory bulb mitral cells, and retinal photoreceptor cells. NeuN can also be detected in neurons in primary cerebellar cultures and in retinoic acid-stimulated P19 embryonal carcinoma cells. Immunohistochemically detectable NeuN protein first appears at developmental timepoints which correspond with the withdrawal of the neuron from the cell cycle and/or with the initiation of terminal differentiation of the neuron. NeuN is a soluble nuclear protein, appears as 3 bands (46-48 x 10(3) M(r)) on immunoblots, and binds to DNA in vitro. The mAb crossreacts immunohistochemically with nervous tissue from rats, chicks, humans, and salamanders. This mAb and the protein recognized by it serve as an excellent marker for neurons in the central and peripheral nervous systems in both the embryo and adult, and the protein may be important in the determination of neuronal phenotype.
Hepatic oval cells (HOC) are a small subpopulation of cells found in the liver when hepatocyte proliferation is inhibited and followed by some type of hepatic injury. HOC can be induced to proliferate using a 2-acetylaminofluorene (2-AAF)/hepatic injury (i.e., CCl4 , partial hepatectomy [PHx]) protocol. These cells are believed to be bipotential, i.e., able to differentiate into hepatocytes or bile ductular cells. In the past, isolation of highly enriched populations of these cells has been difficult. Thy-1 is a cell surface marker used in conjunction with CD34 and lineage-specific markers to identify hematopoietic stem cells. Thy-1 antigen is not normally expressed in adult liver, but is expressed in fetal liver, presumably on the hematopoietic cells. We report herein that HOC express high levels of Thy-1. Immunohistochemistry revealed that the cells expressing Thy-1 were indeed oval cells, because they also expressed α-fetoprotein (AFP), γ-glutamyl transpeptidase (GGT), cytokeratin 19 (CK-19), OC.2, and OV-6, all known markers for oval cell identification. In addition, the Thy-1+ cells were negative for desmin, a marker specific for Ito cells. Using Thy-1 antibody as a new marker for the identification of oval cells, a highly enriched population was obtained. Using flow cytometric methods, we isolated a 95% to 97% pure Thy-1+ oval cell population. Our results indicate that cell sorting using Thy-1 could be an attractive tool for future studies, which would facilitate both in vivo and in vitro studies of HOC.
Central nervous system (CNS) stem cells have become the subject of many laboratories' efforts, presentations, and publications. Yet, in the stem cell world, CNS cells are viewed with skepticism. This is likely due to a dearth of biology (in vivo function) to accompany a flurry of phenomenological and restorative neurology studies. In this article, we compare and contrast the biological knowledge of adult forebrain epidermal growth factor-responsive neural stem cells that has emerged from our laboratories with that of hematopoietic stem cells, using two recent papers in the latter field as specific examples. A comparison of stem cell location, lineage, and repopulation suggests that our understanding of CNS stem cell biology is immature. We conclude that a greater focus on in vivo biology will enhance our knowledge and understanding of CNS stem cells. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 307–314, 1998
Bone marrow stromal cells (BMSC) normally give rise to bone, cartilage, and mesenchymal cells. Recently, bone marrow cells have been shown to have the capacity to differentiate into myocytes, hepatocytes, and glial cells. We now demonstrate that human and mouse BMSC can be induced to differentiate into neural cells under experimental cell culture conditions. BMSC cultured in the presence of EGF or BDNF expressed the protein and mRNA for nestin, a marker of neural precursors. These cultures also expressed glial fibrillary acidic protein (GFAP) and neuron-specific nuclear protein (NeuN). When labeled human or mouse BMSC were cultured with rat fetal mesencephalic or striatal cells, a small proportion of BMSC-derived cells differentiated into neuron-like cells expressing NeuN and glial cells expressing GFAP.
Following a report of skeletal muscle regeneration from bone marrow cells, we investigated whether hepatocytes could also derive in vivo from bone marrow cells. A cohort of lethally irradiated B6D2F1 female mice received whole bone marrow transplants from age-matched male donors and were sacrificed at days 1, 3, 5, and 7 and months 2, 4, and 6 posttransplantation (n = 3 for each time point). Additionally, 2 archival female mice of the same strain who had previously been recipients of 200 male fluorescence-activated cell sorter (FACS)-sorted CD34(+)lin(-) cells were sacrificed 8 months posttransplantation under the same protocol. Fluorescence in situ hybridization (FISH) for the Y-chromosome was performed on liver tissue. Y-positive hepatocytes, up to 2.2% of total hepatocytes, were identified in 1 animal at 7 days posttransplantation and in all animals sacrificed 2 months or longer posttransplantation. Simultaneous FISH for the Y-chromosome and albumin messenger RNA (mRNA) confirmed male-derived cells were mature hepatocytes. These animals had received lethal doses of irradiation at the time of bone marrow transplantation, but this induced no overt, histologically demonstrable, acute hepatic injury, including inflammation, necrosis, oval cell proliferation, or scarring. We conclude that hepatocytes can derive from bone marrow cells after irradiation in the absence of severe acute injury. Also, the small subpopulation of CD34(+)lin(-) bone marrow cells is capable of such hepatic engraftment.
Demyelination is a common pathological finding in human neurological diseases and frequently persists as a result of failure of endogenous repair. Transplanted oligodendrocytes and their precursor cells can (re)myelinate axons, raising the possibility of therapeutic intervention. The migratory capacity of transplanted cells is of key importance in determining the extent of (re)myelination and can, at present, be evaluated only by using invasive and irreversible procedures. We have exploited the transferrin receptor as an efficient intracellular delivery device for magnetic nanoparticles, and transplanted tagged oligodendrocyte progenitor cells into the spinal cord of myelin-deficient rats. Cell migration could be easily detected by using three-dimensional magnetic resonance microscopy, with a close correlation between the areas of contrast enhancement and the achieved extent of myelination. The present results demonstrate that magnetic resonance tracking of transplanted oligodendrocyte progenitors is feasible; this technique has the potential to be easily extended to other neurotransplantation studies involving different precursor cell types.
Neuronal nuclear antigen (NeuN) immunocytochemistry was studied in 15 normal human fetal nervous systems of 8-24 weeks gestation and in four term neonates. Material was derived from products of conception or from autopsy. Antigen retrieval was enhanced for immunocytochemistry by microwave heating of formalin-fixed paraffin sections. NeuN appears highly specific as a marker of neuronal nuclei in human fetal brain. Only rare nuclei are recognized in the germinal matrix. Cerebellar external granule cells are more strongly immunoreactive than postmigratory internal granule cells until 24 weeks gestation; by term most internal and only a few external granule cells are recognized by NeuN antibody. In the cerebrum, some reactive nuclei are demonstrated along radial glial fibers, particularly near the cortical plate. Within the cortical plate, only deep neurons (future layers 4-6) are marked at 19-22 weeks, but by 24 weeks most neurons in the cortical plate exhibit immunoreactivity, though at term some in layer 2 are still non-reactive. Some neurons fail to be recognized by NeuN at all ages: Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells are examples. Despite their common origin in the cerebellar tubercle, basal pontine neurons are strongly reactive even before midgestation, hence NeuN does not predict embryonic origin. Neurons of dorsal root and cranial nerve ganglia are reactive even at 8 weeks. This study of normal fetal central nervous system provides a basis for neuropathological evaluation and as a prelude to applications in cerebral dysgeneses.
Neurotransplantation has been used to explore the development of the central nervous system and for repair of diseased tissue in conditions such as Parkinson's disease. Here, we examine the effects of direct injection into rat brain of human marrow stromal cells (MSCs), a subset of cells from bone marrow that include stem-like precursors for nonhematopoietic tissues. Human MSCs isolated by their adherence to plastic were infused into the corpus striatum. Five to 72 days later, brain sections were examined for the presence of the donor cells. About 20% of the infused cells had engrafted. There was no evidence of an inflammatory response or rejection. The cells had migrated from the injection site along known pathways for migration of neural stem cells to successive layers of the brain. After infusion into the brain, the human MSCs lost their immunoreactivity to antibodies for collagen I. Initially, the human cells continued to stain with antibodies to fibronectin but the region of staining with fibronectin was significantly decreased at 30 and 72 days. The results suggest that MSCs may be useful vehicles for autotransplantation in both cell and gene therapy for a variety of diseases of the central nervous system.
Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with ¹²⁵ I‐labeled anti‐rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated ¹²⁵ I‐labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross‐inhibition of binding of different monoclonal antibodies.
It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat‐stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major ¹²⁵ I‐labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105000. Five IgG‐secreting clones identify the fourth antigen, a heat‐stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross‐inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.
Despite a large number of investigations of embryonic vascular development, in particular in avian embryos, the conditions under which the endothelial and hematopoietic cell lineages emerge remain unknown. As we demonstrate here, both endothelial and hematopoietic cells can be induced by treatment of dissociated quail epiblast with fibroblast growth factors in vitro. These cells aggregate in characteristic blood islands. In long-term culture, the induced endothelial cells gave rise to vascular structures in vitro, i.e. vasculogenesis. No induction was observed in the absence of fibroblast growth factors, and other growth factors like TGF-beta, TGF-alpha and EGF were not capable of inducing blood island formation. Thus, the dissociated quail epiblast provides a remarkably simple test system to investigate cell lineage diversification in higher vertebrates.
The differentiation of adipocytic and osteogenic cells has been investigated in cultures of adult rat marrow stromal cells. Adipocytic differentiation was assessed using morphological criteria, changes in expression of procollagen mRNAs, consistent with a switch from the synthesis of predominantly fibrillar (types I and III) to basement membrane (type IV) collagen, and the induction of expression of aP2, a specific marker for differentiation of adipocytes. Osteogenic differentiation was assessed on the basis of changes in the abundance of the mRNAs for the bone/liver/kidney isozyme of alkaline phosphatase and the induction of mRNAs for bone sialoprotein and osteocalcin. In the presence of foetal calf serum and dexamethasone (10(-8) M) there was always differentiation of both adipocytic and osteogenic cells. When the steroid was present throughout primary and secondary culture the differentiation of osteogenic cells predominated. Conversely, when dexamethasone was present in secondary culture only, the differentiation of adipocytes predominated. When marrow stromal cells were cultured in the presence of dexamethasone in primary culture and dexamethasone and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 10(-8) M) in secondary culture, the differentiation of adipocytes was inhibited whereas the differentiation of osteogenic cells was enhanced, as assessed by an increase in expression of osteocalcin mRNA. The results, therefore, demonstrate an inverse relationship between the differentiation of adipocytic and osteogenic cells in this culture system and are consistent with the possibility that the regulation of adipogenesis and osteogenesis can occur at the level of a common precursor in vivo.
Multipotential CNS stem cells receive and implement instructions governing differentiation to diverse neuronal and glial fates. Exploration of the mechanisms generating the many cell types of the brain depends crucially on markers identifying the stem cell state. We describe a gene whose expression distinguishes the stem cells from the more differentiated cells in the neural tube. This gene was named nestin because it is specifically expressed in neuroepithelial stem cells. The predicted amino acid sequence of the nestin gene product shows that nestin defines a distinct sixth class of intermediate filament protein. These observations extend a model in which transitions in intermediate filament gene expression reflect major steps in the pathway of neural differentiation.
All seven of a set of CD34 monoclonal antibodies that recognize epitopes on an approximately 110 Kd glycoprotein on human hemopoietic progenitor cells also bind to vascular endothelium. Capillaries of most tissues are CD34 positive, as are umbilical artery and, to a lesser extent, vein, but the endothelium of most large vessels and the endothelium of placental sinuses are not. Angioblastoma cells and parafollicular mesenchymal cells in fetal skin are also CD34 positive, as are some stromal elements. An approximately 110 Kd protein can be identified by Western blot analysis with CD34 antibodies in detergent extracts of freshly isolated umbilical vessel endothelial cells, and CD34 mRNA is present in cultured umbilical vein cells as well as other tissues rich in vascular endothelium (breast, placenta). These data indicate that the binding of CD34 antibodies to vascular endothelium is to the CD34 gene product, and not to crossreactive epitopes. Despite the presence of CD34 mRNA in cultured, proliferating endothelial cells, the latter do not bind CD34 antibodies. In addition, CD34 antigen cannot be upregulated by growth factors. We conclude that under these conditions, CD34 protein is downregulated or processed into another form that is unreactive with CD34 antibodies. Electron microscopy of umbilical artery, breast, and kidney capillary vessels reveals that in all three sites, CD34 molecules are concentrated on membrane processes, many of which interdigitate between adjacent endothelial cells. However, well-established endothelial cell contacts with tight junctions are CD34 negative. CD34 may function as an adhesion molecule on both endothelial cells and hematopoietic progenitors.
Hematopoietic stem cells (HSCs) isolated from mouse fetal liver, like adult HSCs, are Thy-1lo Lin- Sca-1+. Donor-derived V gamma 3+ T cells were detected in fetal thymic lobes repopulated in vitro with fetal liver HSCs, but not in those with adult bone marrow HSCs. Single clonogenic fetal HSCs gave rise to thymic progeny that include V gamma 3+, other gamma delta+, and alpha beta+ T cells. No V gamma 3+ T cells were detected in adult thymus injected intrathymically with either fetal or adult HSCs. These results support the hypothesis that only fetal HSCs have the capacity to differentiate into V gamma 3+ T cells in the fetal thymic microenvironment and that the developmental potential of HSCs may change during ontogeny.
The entry of T-lymphocytes into the parenchyma of the central nervous system is a critical early feature in the pathogenesis of many experimental and spontaneously occurring immune-mediated illnesses. The physiological mechanisms controlling this entry have not been elucidated. This study reports that T-cell entry into the rat CNS appears to be primarily dependent upon the activation state of the lymphocytes; T-lymphoblasts enter the CNS (and all other tissues examined) in an apparently random manner while T cells not in blast phase are excluded. Antigen specificity, MHC compatibility, T-cell phenotype, and T-cell receptor gene usage do not appear related to the ability of cells to enter. This study demonstrates that when T-lymphoblasts are introduced into the circulation they rapidly appear in the CNS tissue. Their concentration in the CNS reaches a peak between 9 and 12 hr, and lymphocytes which have entered, exit within 1 to 2 days. Cells capable of reacting with a CNS antigen remain in the tissue or cyclically reenter to initiate inflammation if they are able to recognize their antigen in the correct MHC context. This observation also appears to pertain to the entry of activated T cells into many other tissues, although their concentrations in these non-CNS sites was not quantitated.
Medulloblastoma is a common brain tumor of children. Three differentiated cell types are found in medulloblastomas: neurons, glia, and muscle cells. Because of the presence of multiple differentiated cell types these tumors were named after a postulated cerebellar stem cell, the medulloblast, that would give rise to the differentiated cells found in the tumors. We describe a cell line with the properties expected of the postulated medulloblast. The rat cerebellar cell line ST15A expresses an intermediate filament, nestin, that is characteristic of neuroepithelial stem cells. ST15A cells can differentiate, gaining either neuronal or glial properties. In this paper we show that the same clonal cell can also differentiate into muscle cells. This result suggests that a single neuroectodermal cell can give rise to the different cell types found in medulloblastoma. We also show expression of nestin in human medulloblastoma tissue and in a medulloblastoma-derived cell line. Both the properties of the ST15A cell line and the expression of nestin in medulloblastoma support a neuroectodermal stem cell origin for this childhood tumor.
The cytoskeleton of stromal cells from the adherent layer of human Dexter-type cultures has been studied. It was found that the stress fibers contained actin specific for smooth muscle, mainly the alpha SM actin isoform. The intermediate filaments consisted of vimentin, and there were no desmin filaments. This pattern was similar to that of cultured vascular smooth muscle cells. The detectability of the alpha SM actin isoform is coincident with the appearance of stromal cells in long-term marrow cultures and may provide a useful marker for stromal cells. The potential in vivo cellular counterpart for stromal cells generated in the Dexter-type culture system is discussed.
The pattern of haemoglobin production changes at the embryonic, fetal and postnatal stages of human development, reflecting the expression of different globin genes in both the alpha-like and beta-like gene clusters. Recent studies have identified alterations in the state of DNA methylation and sensitivity to nuclease digestion associated with developmental expression of the globin genes in red blood cell precursors, but the mechanism initiating these changes remains unknown. Despite the screening of large numbers of blood samples from newborn infants, no mutants have been found which affect the timing of these changes (with one possible exception involving a chromosomal translocation), thus necessitating alternative approaches to analysing the cellular basis for the timing of haemoglobin switching. Although many mechanisms are possible, the initiation of the switch from fetal to adult haemoglobin could be regulated essentially either by a developmental clock inherent to haematopoietic stem cells or by an inductive environment, and in an attempt to distinguish between these possibilities, we have transplanted sheep fetal haematopoietic tissue into adult animals. Although previous experiments of this type produced conflicting results, the accumulated results presented here demonstrate that the pattern of haemoglobin production after transplantation is determined largely by the gestational age of the fetal donor cells.
The vocal control nucleus designated HVc (hyperstriatum ventrale, pars caudalis) of adult female canaries expands in response to systemic testosterone administration, which also induces the females to sing in a male-like manner. We became interested in the possibility of neurogenesis as a potential basis for this phenomenon. Intact adult female canaries were injected with [3H]thymidine over a 2-day period. Some birds were given testosterone implants at various times before thymidine. The birds were sacrificed 5 wk after hormone implantation, and their brains were processed for autoradiography. In parallel control experiments, some birds were given implants of cholesterol instead of testosterone. All birds showed considerable numbers of labeled neurons, glia, endothelia, and ventricular zone cells in and around HVc. Ultrastructural analysis confirmed the identity of these labeled neurons. Cholesterol- and testosterone-treated birds had similar neuronal labeling indices, which ranged from 1.8% to 4.0% in HVc. Thus, neurogenesis occurred in these adults independently of exogenous hormone treatment. Conversely, both glial and endothelial proliferation rates were markedly stimulated by exogenous testosterone treatment. We determined the origin of the thymidine-incorporating neurons by sacrificing two thymidine-treated females soon after their thymidine injections, precluding any significant migration of newly labeled cells. Analysis of these brains revealed no cells of neuronal morphology present in HVc but a very heavily labeled ventricular zone overlying HVc. We conclude that neuronal precursors exist in the HVc ventricular zone that incorporate tritiated thymidine during the S phase preceding their mitosis; after division these cells migrate into, and to some extent beyond, HVc. This ventricular zone neurogenesis seems to be a normally occurring phenomenon in intact adult female canaries.
The anti-My-10 mouse monoclonal antibody was raised against the immature human myeloid cell line KG-1a and was selected for nonreactivity with mature human granulocytes. Anti-My-10 immunoprecipitated a KG-1a cell surface protein with an apparent Mr of approximately 115 kD. We describe the binding of this antibody to human hematopoietic cell types and show that My-10 is expressed specifically on immature normal human marrow cells, including hematopoietic progenitor cells. My-10 is also expressed by leukemic marrow cells from a subpopulation of patients. Thus, this antibody allows the identification and purification of hematopoietic progenitor cells from normal human marrow and the subclassification of leukemia.
Four monoclonal antibodies are characterized that have been obtained from a fusion of mouse myeloma P3-NS1/1-Ag4-1 with spleen cells from BALB/c mice immunized with white matter from bovine corpus callosum. The corresponding antigens (O antigens) are designated O1, O2, O3, and O4. The localization of these antigens was investigated by indirect immunofluorescence in cultures of early postnatal mouse cerebellum, cerebrum, spinal cord, optic nerve, and retina. When tested on live cultures none of the O antibodies reacted with the surface of astrocytes, neurons, or fibroblasts, however, all are positive on the surface of oligodendrocytes. The identity of these cells was determined by double-immunolabeling experiments with indpendent cell-type-specific antigenic markers (glial fibrillary acidic protein, tetanus toxin receptors, fibronectin, and galactocerebroside). Antigen O1 is exclusively expressed on galactocerebroside-positive cells, whereas O2, O3, and O4 are expressed on additional cells that are negative for any of the markers tested. None of the O antigens is expressed on the surface of cultured retinal cells. In fresh-frozen sections of adult mouse cerebellum all O antigens are detectable in white matter tracts and in vesicular structures of the granular layer. O2 and O3 antigens are in addition detectable in GFA protein-positive radial fibers in the molecular layer. In fixed cerebellar cultures, where intracellular antigens are accessible, O1, O2, and O3 antibodies label astrocytes in a GFA protein-like pattern. O antigens are expressed in mouse, rat, chicken, and human central nervous systems. O antibodies belong to the IgM immunoglobulin subclass and have been used in complement-dependent cytotoxic elimination of cerebellar oligodendrocytes in culture. At limiting antibody dilutions all processes of oligodendrocytes are preferably lysed over cell bodies.
The restriction of Paneth cell formation to the top of the Paneth cell distribution in the adult was suggested to be due either to the existence of a stem-cell zone or to the influence of a Paneth cell population-density gradient (Bjerkness and Cheng, 1981). To distinguish between the two possible mechanisms, the development of the Paneth cell distribution in neonatal mice (0-10 days old) was studied. If restricted formation were due to the presence of a population-density gradient of Paneth cells, then in neonatal animals, in the absence of a Paneth cell population-density gradient, Paneth cell formation would occur throughout the crypt base. If, on the other hand, restricted formation were due to the presence of a stem-cell zone, and if this mechanism were operative in the newborn, Paneth cell formation in the newborn would be restricted to the region above the stem-cell zone. The position of each Paneth cell within the crypt, and the size of its largest granule, were recorded. On day 0, Paneth cells were present, but crypts were poorly developed and positional assignment was not possible. On day 1, immature crypts developed. All Paneth cells found in immature crypts on day 1 were at the crypt-surface junction (approximately position 5). On day 2, most Paneth cells were at the crypt-surface junction. Thereafter, Paneth cells began to appear at lower positions. On day 3, there were 15 times more Paneth cells in position 5 than in position 1. On day 4, there were still three times more Paneth cells in position 5 than in position 1. With age, the proportion of Paneth cells in position 1 increased while that in position 5 decreased. On day 10 there were more Paneth cells in position 1 than in 5. At all time intervals, granules of Paneth cells in position 1 were significantly larger than those in position 5, indicating that Paneth cells in position 1 were older than those in position 5. It was concluded that in the neonate, before the establishment of a Paneth cell population-density gradient, Paneth cell formation was restricted to positions 5 and above. This supports the existence of a stem-cell zone, not a Paneth cell population-density gradient, as the underlying mechanism of restricted Paneth cell formation in the adult.
The compound 5-azacytidine has been previously shown to convert cells of the rat embryonic fibroblastic cell line, C3H/10T1/2, into myoblasts, adipocytes, and chondrocytes. Rare, resident cells of bone marrow and periosteum, referred to as mesenchymal stem cells, have been shown to differentiate into a number of mesenchymal phenotypes including bone, cartilage, and adipocytes. Rat bone marrow-derived mesenchymal stem cells were exposed to 5-azacytidine beginning 24 h after seeding twice-passaged cells into culture dishes. After an exposure of 24 h, long, multinucleated myotubes were observed in some of the dishes 7-11 days later. Cells containing Sudan black-positive droplets in their cytoplasm were also observed. Thus, culture-propagated rat bone marrow mesenchymal stem cells appear to have the capacity to be induced to differentiate in vitro into myogenic and adipocytic phenotypes, although nonmesenchymal cells (rat brain fibroblasts) cannot be so induced. Taken together, these observations provide support for the suggestion that mesenchymal stem cells in the bone marrow of postnatal organisms may provide a source for myoprogenitor cells which could function in clinically relevant myogenic regeneration.
Stem cell factor (SCF) and its receptor, c-kit, are known to play important roles in hematopoiesis, melanogenesis, and gametogenesis. The biologic effects of the SCF/c-kit system are believed to involve survival, proliferation, and migration of early stem cell progeny. Although SCF and c-kit receptor are widely expressed during normal embryonic development, their expression in the adult is limited.
The expression of SCF and c-kit genes was examined during liver regeneration via the oval cell compartment utilizing partial hepatectomy (PH) combined with the administration of a noncarcinogenic dose of 2-acetylaminofluorene (AAF) for 8 days (AAF/PH model).
Both the ligand and the receptor genes were expressed during the early stages of oval cell proliferation after partial hepatectomy in the AAF/PH model, while neither simple partial hepatectomy nor AAF administration alone induced a noticeable expression of the SCF/c-kit system. The level of SCF mRNA increased within 12 hours after partial hepatectomy and reached a peak around day 4. Thus, the expression of SCF preceded the major expansion of the oval cell compartment. The level of c-kit transcripts gradually increased from the 12-hour time point and stayed elevated until day 11, when a large proportion of the oval cells differentiated into small basophilic hepatocytes. Separation of liver cells at day 3 in the AAF/PH model into parenchymal and nonparenchymal fractions demonstrated that the expression of both SCF and c-kit receptor genes was restricted to the nonparenchymal cells. Furthermore, in situ hybridization revealed that the c-kit transcripts were restricted to oval cells, whereas the SCF transcripts were expressed in both oval cells and Ito cells.
The transcripts for the c-kit receptor are expressed in the early progeny of the hepatic stem cells. The SCF/c-kit system may, possibly in combination with other growth factor/receptor systems, be involved in the early activation of the hepatic stem cells as well as in the expansion and differentiation of oval cells.
As the interest in immunological events occurring in the central nervous system (CNS) has grown in the past quarter century, many investigators have repeatedly confirmed the relative absence of cellular elements of the immune system in the CNS of healthy mammals. This encompasses the lack of a significant number of T and B lymphocytes and low to nonexistent constitutive MHC class I and II antigen expression (Wekerle et al. 1986). Nevertheless, in numerous pathological conditions these cells are readily able to enter the CNS and contribute to salutary or destructive immunological processes. Scientific questions regarding the role and mechanisms of cellular entry are becoming clearer. These questions focus on the following concepts: What is the normal number of immunologically related cells types in the CNS? How do these cells gain access to the CNS under normal and pathological conditions? What important molecular signals and adhesion molecules do they require and how are these “signals” modulated in CNS pathology? In what processes do these hematogenous cells participate? And, how do their arrivals and departures vary in pathological conditions? From a variety of in vivo model systems and in vitro studies, the answers to these and related questions are now beginning to appear.
The receptor tyrosine kinase Flk-1 (ref. 1) is believed to play a pivotal role in endothelial development. Expression of the Flk-1 receptor is restricted to endothelial cells and their embryonic precursors, and is complementary to that of its ligand, vascular endothelial growth factor (VEGF), which is an endothelial-specific mitogen. Highest levels of flk-1 expression are observed during embryonic vasculogenesis and angiogenesis, and during pathological processes associated with neovascularization, such as tumour angiogenesis. Because flk-1 expression can be detected in presumptive mesodermal yolk-sac blood-island progenitors as early as 7.0 days postcoitum, Flk-1 may mark the putative common embryonic endothelial and haematopoietic precursor, the haemangioblast, and thus may also be involved in early haematopoiesis. Here we report the generation of mice deficient in Flk-1 by disruption of the gene using homologous recombination in embryonic stem (ES) cells. Embryos homozygous for this mutation die in utero between 8.5 and 9.5 days post-coitum, as a result of an early defect in the development of haematopoietic and endothelial cells. Yolk-sac blood islands were absent at 7.5 days, organized blood vessels could not be observed in the embryo or yolk sac at any stage, and haematopoietic progenitors were severely reduced. These results indicate that Flk-1 is essential for yolk-sac blood-island formation and vasculogenesis in the mouse embryo.
Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.