Murray Smith

Growth and Differentiation of Embryoid Bodies Derived from Human Embryonic Stem Cells: Effect of Glucose and Basic Fibroblast Growth Factor

Abstract Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.

The induction of neural tube defects by maternal hyperthermia: a comparison of the guinea‐pig and human

In our recent studies on the effects of maternal hyperthermia on the embryonic guinea‐pig, we have demonstrated two ‘teratogenic windows’ at embryonic days 13 and 21 (E13 and E21). E13 encompasses the period of the closure of the neural groove and anterior neuropore, and E21 the commencement of the cortical plate. The approximate equivalent developmental times in the human are E23‐E25 and E49‐E56 respectively. In the guinea‐pig, maternal hyperthermia at E13 results in a high incidence of neural tube defects (NTD), many open, and associated with other defects such as microphthalmia, and scoliosis or kyphosis. The NTD were most common in the developing hindbrain and all demonstrated considerable infoldings of neural tissue, rosettes of neuroepithelial cells, outpocketings of neural tissue and large cystic cavities beneath the defect. In human examples from the Kyoto Human Embryo Collection, 16 had verified hyperthermic insults at E23‐E25 and all had NTD which showed similar deformities to the guinea‐pig. Most embryos with such gross defects are aborted in the early fetal period in both species.

Basic Biology of Pig Fetal Pancreas and Its Use as an Allograft

Diabetes is a chronic disorder of metabolism, which has the potential to result in the development of microvascular complications, such as retinopathy and nephropathy, many years after the onset of the disease process. This is particularly relevant to insulin-dependent diabetes, which affects the young, with blindness and renal failure occurring early in life, rather than at an advanced age, which can occur in non-insulin dependent diabetes. Until insulin dependent diabetes can be prevented, or cured by means of gene transfer, pancreatic transplantation is probably the most likely means at our disposal to normalize blood glucose levels, and thus prevent the occurrence of complications.

The Effects of Hyperthermia on the Fetus

13. Freud, A. (1952) ‘The mutual influences in the development of ego and id.’ Psychoanalytic Study of the Child, 6, 127-168. Bowlby, J. (1969) Attachment and Loss: Vol. I . Attachment. New York: Basic Books. Ainsworth, M. D. S., Blehar, M. C., Water, E., Wall, S. (1978) Patterns of Attachment: a Psychological Study of the Strange Situation. Hillsdale, N.J.: Erlbaum. Grossmann, K., Grossmann, K. E., Spangler, G., Suess, G., Unzner, L. (1985) ‘Maternal sensitivity and newborns’ orientation responses as related to quality of attachment in Northern Germany.’ In Bretherton, I., Waters, E. (Eds.) Growing Points of Attachment Theory and Research. Monographs of the Society for Research in Child Development, 50, 233-256. Goldberg, S., Perrotta, M., Minde, K., Corter, C. (1986) ‘Maternal behaviour and attachment in low birthweight twins and singletons.’ Child Development, 57, 34-44. Egeland, B., Farber, E. A. (1984) ‘Infantmother attachment: factors related to its development and changes over time.’ Child Development, 55, 753-771. Erickson, M. F., Sroufe, L. A., Egeland, B. (1985) ‘The relationship between quality of attachment and behaviour problem,s in preschool in a high-risk sample. In Bretherton, I., Waters, E. (Eds.) Growing Points of Attachment Theory and Research. Monographs of the Society for Research in Child Development, 50, 147-166. Main, M., Kaplan, N., Cassidy, J. (1985) ‘Security in infancy, childhood, and adulthood: a move to the level of representation.’ In Bretherton, I., Waters, E. (Eds.) Monographs of the Society for Research in Child Development, 50, 66-104. Klaus, M., Kennell, J. (1982) Parent-Infant Bonding, 2nd Edn. St. Louis: C. V. Mosby. Myers, B. (1984) ‘Mother-infant bonding: the status of the critical period hypothesis.’ Developmental Review, 4, 240-274. Klaus, M. H., Jerauld, R., Kreger, N. C.,Steffa, M., Kennell, J. H. (1972) ‘Maternal attachment: importance of the first postpartum days.’ New England Journal of Medicine, 286, 460-463. Hwang, C. P. (1981) ‘Aspects of the motherinfant relationship during nursing, 1 and 6 weeks after early extended post-partum contact.’ Early Human Development, 5,

In vitro Dedifferentiation of Fetal Porcine Pancreatic Tissue prior to Transplantation as Islet-Like Cell Clusters

The fetal porcine pancreas under experimental conditions can be transplanted in the form of explants or islet-like cell clusters (ICCs) to normalize blood glucose levels in diabetic recipients. ICCs are released from the collagenase-digested pancreas and require a 4- to 5-day culture period for their complete formation. In order to maximize insulin producing β cell differentiation following transplantation, an understanding of ICC development is essential to utilize this alternative treatment for type 1 diabetes. In this study a role is proposed for exocrine cells in the generation of the multipotent pancreatic precursor cells during the culture period. Acinar cells undergo dedifferentiation during the initial stages of the cultureperiod into multipotent pancreatic precusor cells, previously called protodifferentiated cells. The progressive loss of exocrine differentiation appears to involve rapid degranulation of zymogen granules by exocytosis and loss of the prominent secretory apparatus. These processes occur in parallel with a significant reduction in the expression of lipase in the period from day 0 to day 5 and simultaneously there is an increase in the epithelioid/ductal cell marker, cytokeratin 20. Using proliferating cell nuclear antigen, cell proliferation during the culture period does not appear to account for the increase in epithelioid/ductal cells. Further the rates of apoptosis and necrosis which were identified using the TUNEL technique and propidium iodide, respectively, do not appear to account for the reduction in exocrine cell numbers. Exocrine cell dedifferentiation appears to increase the pool of protodifferentiated cells which have the potential to develop into the insulin-producing β-cell population following transplantation into the diabetic recipient

Heat shock proteins and their role in early mammalian development

Elevated body temperature can result from many agents in the natural environment, such as fever, hot weather and heavy exercise. In our modern living conditions additional sources of induced hyperthermia including hot baths, saunas, drugs, electromagnetic radiation and ultrasound have been introduced. Hyperthermia during pregnancy has been shown to cause a wide spectrun of effects in art species studied, including humans, the outcome depending on the dose of heat absorbed by the mother and embryo and the stage of enbryonic or fetal development when exposed. The dose of heat is the product of the elevation of temperature above normal and the duration of the elevation. In relatively uncontrolled natural environmental exposures, embryonic death and resorption or abortion are probably the most common outcome. In less severe exposures (smaller doses) major or minor developmental defects can result and the central nervous system appears to be a major target for its effects. Heat damage to embryos appears to be by apoptotic and other forms of cell death in organs at critical stages of development. Over many millennia all living orgaisms appear to have developed protective mechanisms against excess heat, known collectively as the heat shock response. This response has been studied intensively over the last 20 years and its mechanisms of protection are now becoming more clearly defined. Exposures to heat (and a number of other toxic agents) trigger the heat shock response which is characterized by abrupt suspension in the normal protein synthesis and the concurrent induction of heat shock genes (hsp) and the synthesis of a set of protein families known collectively as the heat shock proteins (HSP). The hsp ape known to be involved in the response in embryos, each has at least two copies, one which appears to have functions in the normal embryonic development (cognate) and another which is induced at a certain dose of heat (induced) and which can offer some protection against damage for some time after the initiating dose. Most cognate HSP can normally be found in embryos at all stages of development. At certain critical, early stages of organ formation increased activity of one or more of the hsp families can be identified at the site of the organ analogue. The inducible HSP are usually undetectable during normal development and generally become inducible at these critical inductive stages of organ development, implying a protective function for that process. Excess heat is known to cause denaturation of proteins. Each of the known HSP families appears to protect cells through their chaperone functions in which they bind to adhesive sites on newly synthesized or heat damaged and partially unfolded structural and functional proteins. This prevents the formation of function-less aggregates. The damaged proteins are then either presented for degradation or are reconstituted by orderly disengagement from the chaperone protein. The molecular mechanisms of initiating and regulating the response are now becoming more clearly defined. Trigger mechanisms include release of prostaglandin Al which can be modulated by glucocorticoids and nonsteroidal anti-inflammatory agents. A heat shock factor (HSF) binds to the heat shock element (hse) on the gene sequence and initiates the hsp response. The signal induction pathway involves mitogen activated proteins (MAP) and stress activated proteins (SAP) which are regulated by phosphorylation. Signals are amplified by kinase cascades while they are being transmitted to the nucleus. Activated MAP and SAP kinases regulate the process by phosphorylation of proteins including transcription factors, HSP, other protein kinases and phosphorylases, growth factor receptors and cytoskeletal proteins. Although this research has defined some pathways indicating how and why heat can cause some defects, a means of preventing them has not yet emerged.

Interference with neural crest migration by maternal hyperthermia as a cause of embryonic death due to heart failure

Maternal hyperthermia has been demonstrated to be a teratogen in every animal species studied, and a minimum core temperature rise of 2.5 degrees C can produce a number of developmental defects. However, numerous embryos fail to survive to term. In the guinea-pig, heating the embryos prior to neural tube closure induces significant neural tube defects, but all embryos die within 20 days of heating. A number have aberrant cardiac development and many show spectacular pericardial effusions and congestion in the peripheral circulation. We suggest that maternal hyperthermia has interfered with neural crest migration which is a major component in the induction of these changes.

Three-dimensional reconstruction of tissue using computer-generated images

In the study of brain ventricles for both teaching and research, it is often of considerable advantage to graphically display the reconstructed shape of the specimen. This paper describes the making of two-dimensional serial cross-sections and their storage for subsequent manipulation and display on a personal computer; the three-dimensional (3D) reconstructions may have hidden lines removed and various parts coloured for definition of areas of interest, for example the density and position of pyknotic (dead) nuclei. Current research involved the investigation of the effects of maternal hyperthermia on early embryonic brains. The 3D reconstructions were found ideal for understanding the temporal changes occurring in embryonic brains subjected to defined maternal heat stresses. The method involved 4 X 4 matrices using homogenous coordinate theory, being the most ideal and allowing a constant mechanism for all transformations. Total time from examination of sections to obtaining an accurate printed 3D reconstruction is approximately 1 h if 22 sections are used.

Effects of Maternal Hyperthermia on the Developing Guinea-pig Eye

Abstract Maternal hyperthermia of short duration had significant short and long term effects on the developing guinea‐pig eye at embryonic days 19, 21 and 23. A marked regionalisation of the heat‐induced effects was observed in the developing retinae, but the lens defects were most obvious following heat at E21 and E23. Bilateral anterior pole cataracts, bipolar zonular cataracts and microphthalmia have been produced following maternal heat stress. It is suggested that these are determined by the cellular events initiated in the developing eye following the teratogenic insult.

Regulation of pancreatic cell differentiation and morphogenesis

Abstract: Organogenesis requires tissue interactions to initiate the cascade of inductive and repressive signals necessary for normal organ development. Tissue interactions initiate the pancreatic lineage within the primitive foregut endodermal epithelium and continue to direct the morphogenesis and differentiation of the endocrine, exocrine and ductal portions of the pancreas. An understanding of the mechanisms controlling pancreatic growth would enable the development of alternative therapies for diseases such as type 1 diabetes.