Cells: from Robert Hooke to Cell Therapy – a 350 year journey

In 1665 Robert Hooke published Micrographia, the world's first fully-illustrated book of microscopy, and revealed to his readers a completely new world. Along with images of insect, mineral and plant specimens he included a section of cork, describing the tiny box-like structures he observed as 'cells'. Although he did not understand their biological function fully, he attributed to them some of the properties of cork such as buoyancy. 

In Micrographia, Hooke had two goals: to present his observations, and to persuade his readers that scientific research had the potential to improve people’s lives. Hooke made a point of linking his observations with his readers’ experiences of everyday life – for example, including an enormous image of a louse clinging to a human hair, rather than simply showing the insect alone on the page. By investigating the microscopic world, Hooke hoped to understand the mechanisms and structures of nature and perhaps make use of them in his own instruments and inventions. This talk will show that Hooke’s microscopic research underpinned his theories of the natural world, including his conviction that fossils were the remains of once-living plants or animals, a theory that would only be widely accepted over a century after Hooke’s death.

The transition from single cell species to multicellular organisms led to organ and tissue development, which has resulted in the emergence of specialized cells that self-renew as well as differentiate, called stem cells. Following embryonic development, most tissues and organs are continuously regenerated from tissue/organ specific stem cells. In most tissues only the primitive stem cells self-renew. Within a tissue, stem cells may be diverse, and subject to stem cell competition for their niche. Both germline stem cell and somatic stem cell competition in species as diverse as colonial protochordates and humans have been documented. Stem cell isolation and transplantation is the basis for regenerative medicine. Self-renewal is dangerous, and therefore strictly regulated. Poorly regulated self-renewal can lead to the genesis of cancer stem cells, the only self-renewing cells in the tumor. Dr Weissman and colleagues have followed the progression from hematopoietic stem cells (HSCs) to myelogenous leukemias. It was found that the preleukemic clones progress at the stage of HSCs, from which emerge “leukemia” stem cell at an oligolineage downstream stage that has evaded programmed cell death [PCD] and programmed cell removal [PrCR], that self-renews.  In early chronic myeloid leukemia, bcr-abl+ HSC clones outcompete normal HSCs. The transition from the chronic phase to myeloid blast crisis results in the leukemia stem cells appearing in the granulocyte-macrophage progenitor (GMP) stage. While there are many ways to defeat PCD and senescence, there appears to be one dominant method to avoid PrCR—the expression of cell surface “don’t eat me” CD47, the ligand for macrophage SIRPα. All cancers tested express CD47 to overcome expression of “eat me” signals such as calreticulin. Antibodies that block the CD47–SIRPα interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo. All tested human cancers are susceptible to phagocytosis in the presence of anti-CD47 blocking antibodies, which are in clinical trials.

The central nervous system (CNS) is the most complex of tissues, with hundreds of types of neurons and glia patterned into unique regions, connected through intricate, complex circuits. Developmental studies have shown that the initial plate of neuroepithelial cells is regionally patterned by morphogenic signals. This areal identity is then interpreted by neural stem cells and more restricted precursor cells, collectively called neural progenitor cells (NPCs), to produce the diverse cells of each CNS region. Progeny arise from NPCs on a defined schedule, and migrate to their final position. The birthdate of each cell type is precise for a given species, but varies widely, e.g. from mice to men.  

Studies of these developmental events have revealed numerous classes of NPCs. Some are present just through the developmental period, others are retained in active stem cell zones throughout life. Notably, in the hippocampal dentate gyrus, stem cells continually make new neurons that contribute to memory formation. Adult progenitors capable of producing oligodendrocytes and astrocytes are also present widely in the CNS.

Neurodegenerative diseases typically attack specific CNS populations, for example, the hippocampal stem cell system in Alzheimer’s Disease, and midbrain dopaminergic neurons in Parkinson’s disease. Traumatic damage can cause loss of numerous cell types. Human NPCs are being harnessed to replace the lost cells and are already in clinical trials. In addition, the fact that progenitor cells are present in the adult CNS offers the exciting opportunity for stimulating endogenous reparative processes, reawakening the inner salamander.

Nuclear transfer involves the transfer of the nucleus from a donor cell into an egg from which the chromosomes have been removed was considered first as a means of assessing changes during development in the ability of the nucleus to control development. In 1996 a nucleus from an adult cell controlled development to term of Dolly the sheep. She was the first adult clone derived by transfer of a nucleus from an adult cell. The new procedure has been used to target precise genetic changes into livestock; however, the greatest inheritance of the Dolly experiment was to make biologists think differently. If unknown factors in the recipient egg could “reprogramme” the nucleus to a stage very early in development then there must be other ways of making similar changes. Within 10 years two laboratories showed that the introduction of selected proteins changed some of the cells to stem cells equivalent to those derived from an embryo. This ability is providing revolutionary new opportunities in research and cell therapy.

The old is going destiny for us. However mature differentiated cells making up our body can be rejuvenated to embryo-like fate called pluripotency which is an ability to differentiate into all cell types by enforced expression of defined transcription factors. The discovery of this induced pluripotent stem cell (iPSC) technology has opened up unprecedented opportunities in regenerative medicine, disease modeling and drug discovery. In this talk, Dr Takahashi will introduce the process leading to iPSC technology, applications and future perspectives of human iPSCs. And, also show that how iPSC technology has evolved along the way.

Cellular differentiation and lineage commitment are considered robust and irreversible processes during development. Challenging this view, we found that expression of only three neural lineage-specific transcription factors Ascl1, Myt1l, and Brn2 could directly convert mouse fibroblasts into functional in vitro. These induced neuronal (iN) cells expressed multiple neuron-specific proteins, generated action potentials, and formed functional synapses. Thus, iN cells are bona fide functional neurons. Addition of a fourth transcription factor Neurod1 also enabled reprogramming of human fibroblasts.

Completely unexpectedly, the iN cell reprogramming process is substantially more efficient and faster than reprogramming to pluripotent stem cells. By investigating the mechanism of action of the transcription factors involved, we found a molecular explanation for this discrepancy. We found that Ascl1, one of the key driving iN cell transcription factors, is not only a pioneering factor in the sense that it can bind nucleosomal DNA in the fibroblast chromatin context, but we observed it  bound to its physiological targets two days after infection in mouse fibroblasts as defined as targets in neural stem and progenitor cells. The three main iPS cell reprogramming factors Oct4, Sox2, and Klf4, on the other hand, have been shown to bind nucleosomal DNA but mostly bind ectopic genomic sites that are typically not bound by them in ES cells. Thus, the specific properties of the transcription factors used for reprogramming and their interaction with the chromatin appear to determine the reprogramming dynamics and efficiency between reprogramming systems. We also found that expression of neuronal lineage determining factors in human ES and iPS cells yields functionally mature neurons of unprecedentedly speed. These “ES-iN cells” are in quality almost equivalent to a primary neuronal culture and we have shown to be useful to study synaptic phenotypes of disease-associated mutations.

Derivation of many different cell types from human pluripotent stem cells (embryonic stem cells or HESCs and induced pluripotent stem cells or hiPS cells) is an area of growing interest both for potential cell therapy and as a platform for drug discovery and toxicity. Most particularly, the recent availability of methods to introduce specific disease mutations into human pluripotent stem cells and/or to derive these cells as hiPS cells by reprogramming from any patient of choice, are creating unprecedented opportunities to create disease models “ in a dish” and study ways to treat it or slow down its rate of development. Understanding the underlying developmental mechanisms that control differentiation of pluripotent cells to their derivatives and mimicking these in defined culture conditions in vitro is now essential for moving the field forward. Professor Mummery and colleagues have used these methods to produce isogenic pairs of hiPSC lines to compare diseased and corresponding control cardiomyocytes and vascular endothelial cells and identify disease related phenotypes and mechanisms. The use of isogenic pairs has proved crucial since variability between “healthy control” hiPSC lines is often greater than the difference between a diseased cells and its isogenic control. Drug responses of hESC-derived cardiomyocytes to a variety of cardiac and non-cardiac drugs were examined and shown that iPSC derived cardiomyocytes with mutations in ion channel genes can accurately predict changes in cardiac electrical properties and reveal drug sensitivities also observed in patients.