Stem cells

Stem cells are undifferentiated cells with the ability to divide through mitosis to produce more stem cells (self-renewal) and that can differentiate into specialized cells. 

The relevance of stem cells in biomedical research was reflected in the decision to award Sir John B. Gurdon (Dippenhall, United Kingdom, 1933) and Professor Shinya Yamanaka (Osaka, Japan, 1962) the Nobel Prize of Medicine in 2012 for their findings in this research topic.

 

 Taken from the Official Website of the Nobel Prize.

Stem cells are widely studied, due to their potential therapeutic use and for their inherent interest. As explained during the lesson, stem cell research is now focused on regenerative medicine as well as on the treatment of a wide variety of diseases, including cancer, Parkinson disease, multiple sclerosis, and spinal cord injuries, among others. However, most of the clinical trials using stem cells as therapy are still in early clinical phases. The following video explains what stem cells are and how scientists are harnessing their medical potential:

 

Classification of stem cells according to developmental potentials

• Totipotent stem cells derive from the fusion of a sperm cell with an egg cell (zygote) or from the first few divisions of the fertilized egg (morula). Totipotent stem cells can give rise to an entire functional organism or to any cell type of the body, including the extraembryonic tissues (i.e. placenta, umbilical cord, amniotic sac).
• Pluripotent stem cells derive from the inner cell mass of the blastocyst. Pluripotent stem cells can differentiate into nearly all cell types of the three embryonic germ layers (endoderm, mesoderm, ectoderm), but not into an entire functional organism.
• Multipotent stem cells can develop into more than one cell type, but only those of a closely related family of cells (i.e. multipotent blood stem cells are capable of differentiating into erythrocytes, lymphocytes or platelets).
• Unipotent stem cells can produce only one cell type, their own, but have the property of self-renewal, which distinguishes them from non-stem cells.

 Taken from Eckfeldt et al. Nature Reviews Molecular Cell Biology 2005;6:726-737

Classification of stem cells according to their origin

• Embryonic stem cells derive from the embryonic stage of morula (totipotent stem cells) or from the inner cell mass blastocyst (pluripotent stem cells). Due to their high developmental potential, embryonic stem cells can give rise to teratomas, that are tumors containing differentiated elements of all three embryonic germ layers. Isolation of human embryonic stem cells results in the destruction of the fertilized embryo, which raises ethical issues.
• Adult or somatic stem cells originate from adult tissues and they have the ability to divide or self-renew indefinitely and generate the cell types from the tissue from which they originate. This type of cells are multipotent stem cells.
• Induced pluripotent stem cells (iPS): Professor Shinya Yamanaka (University of Kyoto, Japan) reprogrammed, for the first time, murine skin fibroblasts into multipotent stem cells that could develop into all cell types of an adult mouse by introducing four factors Oct3/4, Sox2, c-Myc and Klf4. He named the cells induced pluripotent stem cells (iPS) and these results were published in the prestigious Cell journal in 2006. In the present video, you can see the methods used to obtain iPS cells:

 

You will have to know the:

• Classification and characteristics of stem cells according to their developmental potential (totipotent, pluripotent, multipotent and unipotent stem cells).
• Classification and characteristics of stem cells according to their origin (embryonic, adult and inducible pluripotent stem cells).
• Genes implicated in the induction of iPS cells.

Legal and Forensic Medicine

What is Forensic Medicine?

Legal and Forensic Medicine, as we conceive it nowadays, emerged in the XVI century when medical intervention was required for legal proceedings. Forensic medicine can be defined as medicinal procedures that are applied to judicial decisions. By request, a doctor must report, declare or inform in accordance with existing criminal law. Judges at any moment could ask for help from a doctor and the doctor is obliged to provide this help. The doctor´s declaration may determine the judicial decision.

What does a forensic doctor do?

The activity carried out by forensic doctors is public and it is required by judges, public prosecutors’ offices and justice courts.

Some tasks performed by forensic physicians may be found in the following video:

Legal and Forensic Medicine is a broad speciality encompassing a wide number of SUBDISCIPLINES: 

Forensic Toxicology: It can be applied to a living person or to a corpse. It is characterized by the need of an accurate and quantitative result. It is required when a toxic substance may have produced a psychological alteration or when a crime is committed under its influence. Furthermore, it is also necessary when a death may be due to intoxication, or when a toxic is found in a body.

Dactyloscopy: Fingerprinting deals with the study, classification, register and recuperation of the printings of distal hand phalanges. This subspecialty is the best known applied science for identification of individuals.

There are other important prints:

Forensic Genetics: It consists of the analysis of DNA with forensic aims, among them: biological evidence of kinship, forensic biology, body identification (e.g.: mass disasters, identification of corpses, etc.) and evolution studies.

Forensic Thanatology/Pathology: This subspeciality studies the human death phenomenon. It applies the scientific method to forensic techniques in order to confirm a death and establish the cause of death. The autopsyis a post-mortem surgical procedure aimed to establish the causes of the death and to determine the presence of diseases or injuries.

In the following video an autopsy performed to find out what caused the death is shown (It contains images that may be disturbing):

The possibility to perform virtual autopsies is one of the recent breakthroughs in medicine. With techniques such as computed tomography and magnetic resonance imaging, a virtual "bloodless" autopsy can be performed on a potential victim of a crime. It can be observed in the following video:

Forensic Anthropology: It deals with the study of bone remains and other marks or lesions in order to identify a corpse: the cause of death, date, age, gender, race and anthropometric characteristics, providing the maximum information to the police to help for the identification of the individual. This process starts when the police finds an unidentified corpse skeletonized or in an advanced state of putrefaction.

Forensic Psychiatry: Aims to clarify those cases that need a particular consideration due to a special mental condition of the person involved. Nowadays, Psychiatry and Law are closely linked when a crime has been committed by a person with a mental disorder.

In Spain, legal and forensic Medicine is an official service depending on regional “Comunidades Autónomas” (in those in which the governance has been transferred) and in other cases from the Instituto Nacional de Toxicología y Ciencias Forenses (INTCF). The INTCF is a technical body under the Ministry of Justice, whose mission is to assist the administration of justice and contribute to the unity of scientific criteria and the quality of analytical competence, and the development of forensic science. His organization and supervision is under the control of the Ministry of Justice. It is based in Madrid and its scope extends to the entire national territory.

1. What do we understand by legal and forensic medicine?

2. Know the different subdisciplines of legal and forensic medicine.

3. Medicolegal autopsy: What is it and when is it performed?

4. Familiarize yourself with the different types of toxicological tests.

New Perspectives in Oncology Research

Cancer is the uncontrolled growth and spread of cells. There are many different types of cancers that affect any part of the body. According to the World Health Organization, lung, stomach, liver, colon and breast cancer cause most of the cancer deaths each year.

Tumours can be classified as follows:

• Carcinomas arise from the cells that cover external and internal body surfaces.
• Sarcomas are cancers arising from cells found in the supporting tissues of the body.
• Lymphomas arise in tissues of the body's immune system.
• Leukemias are cancers of the immature blood cells.

A variety of technical names to distinguish the many different types of cancers are used by physicians and scientists:

The transformation from a normal cell into a tumour cell is a multistage process, typically a progression from a pre-cancerous lesion to malignant tumours. The hallmark characteristics in tumour development are:

• Uncontrolled cell proliferation
• Loss of apoptosis
• Angiogenesis
• Tissue invasion and metastasis

Changes in tumoural cells are the result of the interaction between an individual's genetic factors and external agents, including physical, chemical and biological carcinogens. Ageing is also another fundamental factor for the development of cancer and tobacco use is the largest preventable cause of cancer in the world causing 22% of cancer deaths.

Taken from Bhatia and Sklar. Nat Rev Cancer 2002

Cancer cells often invade surrounding tissue and can metastasize to distant sites. Invasion refers to the direct migration and penetration by cancer cells into neighbouring tissues. Metastasis refers to the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then invade normal tissues elsewhere in the body.

A significant proportion of cancers can be cured by a careful selection of one or more therapeutic approaches, such as surgery, radiotherapy, chemotherapy, and new treatments (targeted therapy: inhibitors of angiogenic factors, biological therapies or vaccines):

The tumor markers are substances produced by tumor cells and/or by other cells of the body in response to cancer. Some are related to a single type of cancer, although most of them are associated with two or more cancers. Biomarkers can be used with diagnostic aims, to detect cancer in early stages, as well as with therapeutic purposes, using them to plan an appropriate therapy regimen or for follow-up to evaluate the patient's response to treatment. However, so far no specific biomarker with enough sensitivity and specificity has been identified.

 

Emerging technologies provide the means by which new and single cancer biomarkers could be discovered:

A review of causes and treatment options for cancer can be observed in the following video:

1. Learn the hallmark characteristics of tumour development.
2. Familizarize yourself with the concept of tumour marker. 
3. Study the process of angiogenesis and metastasis as well as the main molecules involved.
4. Understand the concept of circulating tumour cells and minimal residual disease.
5. Be able to identify the main treatment approaches used in oncology.

Gene Therapy: Present and Future

Gene therapy is a technique using vectors, either viral or non viral, for introducing therapeutic genetic material into target cells to solve a specific problem.

The main events that led to the development of gene therapy include: 

• Discovery of DNA as the keeper of genetic information.
• Development of PCR.
• Sequentiation of the human genome.
• Finding of restriction enzymes.

In September 1999, 18-year-old Jesse Gelsinger took part in a gene-therapy clinical trial. The adverse patient reaction to an adenovirus vector during the clinical safety trial led to the realization that the failure to understand the biology of vector interactions with the human immune system could have fatal consequences. 

In April 2000, a paper published in Science marked the highest point in the history of gene therapy. It described the first gene therapy success in which three children were cured of a fatal immunodeficiency disorder, but this therapy has subsequently caused a leukaemia-like disease in 2 of the 11 patients who have been treated.

The efficiency of gene therapy mainly resides in vectors. Intense efforts have been pointed to understand the molecular basis of how viruses and viral vectors interact with the host. In this sense, an ideal vector should have the following features:

• Do not trigger immune response.
• Easy to prepare.   
• Enough capacity to harbour large genes.
• Act both in proliferating and in resting cells.
• Easily dirigible to the target cell.
• Survival during large periods of time in the infected/transfected cells.
• Have regulatory elements to correct gene expression.

Vectors are classified in viral and non-viral:

1. Viral vectors: Viral vectors were the first to be used, given its high efficacy as vehicles for nucleic acids. Viruses are composed of DNA or RNA surrounded by a protein capsid, and in some cases, a lipoprotein envelope. There are five main classes of clinically applicable viral vectors: retrovirus, lentivirus, herpes simplex virus-1, adenovirus and adeno-associated virus.

Taken from Thomas, Ehrhardt and Kay. Nat Rev Genet 2003.

 Steps in gene therapy using an adenovirus vector:

2. Non-viral vectors: This type of vectors represents an attempt to mimic the virus functions as transferring vehicles using synthetic systems, but reducing the adverse characteristics of viruses. Efficacy of infection is much lower as it has to overcome both external and internal barriers. Liposomes and naked DNA are the main useful types with the advantage of unlimited length of the genomic material that can be introduced. However, they are still less efficient than viral vectors.

A summary of the extra- and intracellular barriers faced by non-viral gene therapies following systematic delivery can be observed below:

Taken from Miyata et al. Chem Soc Rev 2012 and McCrudden and McCarthy. Cancer Gene Therapy – Key Biological Concepts in the Design of Multifunctional Non-Viral Delivery  Systems. Gene Therapy - Tools and Potential Applications. ISBN 978-953-51-1014-9.

Nowadays, the discovery of interference RNA has opened a new field concerning the control of gene expression.

Target cells are selected depending on the tissue in which the gene should be expressed. They must also be cells with a long half-life being important to consider the rate of division of the cells. Best target cells are hematopoietic stem cells but lymphocytes, respiratory epithelium, hepatocytes, fibroblasts and muscle cells are highly targeted in gene therapy.

In the following videos, the gene therapy procedures to Parkinson and tumoural diseases are explained:

1. Definition of gene therapy and main developments of the field.
2. Know the difference between in vivo and ex vivo.
3. Study the characteristics of an ideal vector.
4. Know the classification of vectors and types of genetic information inserted.
5. Familiarize yourself with the main applications used nowadays.

New Technologies in Neurosciences

Neuroscience encompasses the study of the nervous system. It not only involves the study of its anatomy and physiology, but also the diagnosis and treatment of the pathologies in relation with the nervous system.

Thanks to past and present research, neuroscience has evolved from studying the anatomy and physiology of the brain to finding cures for complex neurological disorders. The genetic variation and non-Mendelian inheritance create a challenge in understanding neurological disorders that disrupt the normal processes of the brain such as Alzheimer’s disease. The following video shows the evolution of Alzheimer’s disease:

Due to advances in neuroscience the horizon looks brighter. Molecular biology has played an important role in understanding the genetic characteristics behind neurological disorders, but also imaging technologies are now contributing to a better understanding of the human brain. The Human Brain Project and the Brain Activity Map Project seek to integrate everything we know about the brain into massive databases and detailed computer models.

In this sense, in order to find a cure, researchers use different techniques in a multidisciplinary approach that spans from radiology to molecular biology. The Human Connectome Project aims to provide an unparalleled compilation of neural data as well as an interface to navigate across these data to achieve conclusions about the living human brain.

By understanding how the human brain works, we can effectively identify and target the affected areas that cause neurological diseases:

• The brain is an enormously complicated system of interconnected cells. Johnson and Wu suggest that the human brain has 10¹² neurons with 10¹⁵ synapses. The magnitude of 10¹⁵ synapses is about 222 times greater than the distance from Earth to Pluto in meters.  

• Sleep has an important role in memory. Recent studies have shown that presentation of smells or sounds that accompanied learning can enhance procedural and episodic memories when re-presented in sleep. Welberg L. Nat Rev Neurosci 2013, 14: 737. 

• In the 18th of October issue of Science a mechanistic explanation for how sleep, in addition to its well-described effects on memory consolidation, facilitates the clearance of potentially neurotoxic waste products that accumulate during wakefulness was published. Xie L. Science 2013, 342:373.

• Chronic pain is estimated to affect over one-quarter of the world's population, and presents a considerable therapeutic challenge. The aim is to understand the risk factors and mechanisms that underlie chronic pain to develop effective and non-addictive treatments for this condition.

• The Nobel Prize in Physiology or Medicine 2014 was awarded with one half to John O'Keefe and the other half jointly to May-Britt Moser and Edvard I. Moser "for their discoveries of cells that constitute a positioning system in the brain"

• Be able to identify different techniques applied to neuroscience research.
• Know the main imaging and molecular techniques used in neurosciences.
• Study new markers and treatments for neurological diseases.
• Know about surgical procedures in neuroscience.
• Concept of optogenetic technique. 

"Omics" technologies

The complete sequencing of the human genome has introduced us in a new era of research methodology referred as omics technologies. This term comprises high-throughput experimental technologies characterized by automation, miniaturized assays and large-scale data analysis. These technologies are increasingly generating massive and complex genomic data sets so that interpreting functional consequences of millions of discovered genetic variants is one of the biggest challenges. Over the past few years omics technologies have revolutionized research projects.

There are five major types of high-throughput measurements that are commonly performed:

• Genomic: analysis of the DNA sequence (i.e., SNP).
• Transcriptomic: analysis of transcribed RNA (i.e., the simultaneous measurement of gene expression values in a cell or tissue type).
• Proteomic: determination of proteins present in a sample.
• Metabolomic:  identification and quantification of all metabolites in a sample.
• miRNAomics: regulatory mechanisms underlying control of transcription.

Schematic representation of omics technologies, their corresponding analysis targets, and assessment methods. Taken from Wu RD et al. JDR 2011; 90:561-572.

Below is a list of the various types of information obtained from some of the omics technologies (adapted from Sawyers CL. Nature 2008; 452: 548-52):

• DNA copy-number assessment: comparative genome hybridization to DNA microarrays.
• Mutation screening: DNA sequencing,  mass-spectrometry-based genotyping, mutation-specific PCR.
• Gene-expression profiling: microarrays, multiplex PCR, microRNA-expression profiling. 
• Proteomic profiling: mass spectrometry, phosphoproteomic profiling, mass spectrometry after immunoprecipitation with specific antibodies. 
• Metabolomic profiling: mass spectrometry.

Tissue microarrays (TMAs) are an ideal platform for validating the results obtained from large-scale transcriptomic and proteomic studies. The most commonly performed assays on tissue microarrays are immunohistochemistry for protein expression, in situ hybridization for RNA expression, and fluorescence in situ hybridization for DNA copy number.

TMAs accrue value over time and allow investigators to build u

p IHC

profiles for individual tumours, as demonstrated by the expression of six different biomarkers in a single tumour core. Taken from Brennan DJ et al. Cancer Genomics Proteomics 2007; 4:121-134.

2-D eletrophoresis is an important step in proteomics analysis. In the following video you can learn more about how to prepare 2-D gels:

This video shows microarray technology and applications:

1. Study the main omics technologies and identify their differences.
2. Know the methodology used by each omic subdiscipline.
3. Advantages and limitations related to omics technologies.
4. Be able to relate the omics methodologies with the distint type of research.
5. Familiarize yourself with the diverse types of analysis in proteomics.
6. Know what epigenomics referes to.

 

 

 

Basic molecular biology in biomedical research

Milestones of molecular biology

The major advances in genetic engineering have occured during the last 6 decades, but the history of molecular biology started at the end of the XIX century with the research of Darwin and Mendel. On his book "On The Origin of Species", Charles Darwin explained that heredity is the mechanism that perpetuates variations, since the traits in offspring result from a blending of the traits of each parent. The Austrian friar Gregor Mendel conceived the idea of heredity units, which he called "factors" (later known as "genes"), by studying Pisum sativum, or the common pea plant. Mendel proposed that "factors" normally occur in pairs and these two "particles" of the factors (later known as "alleles") segregate from each other in the formation of gametes. Therefore, Mendel stated that each individual inherits two "particles" for each trait, one from each parent.  In 1869, Friedrich Miescher first isolated what he called "nuclein" (later known as "deoxyribonucleic acid" or "DNA" inside the nuclei of human white blood cells. 

 

In 1953, Dr. James Watson and Dr. Francis Crick deduced the three dimensional, double-helical model for the structure of DNA, based on some crucially important X-ray chrystallography work of Dr. Rosalind Franklin and Dr. Maurice Wilkins. Watson, Crick and Wilkins received the Nobel Prize in Medicine for the double-helix model of DNA in 1962, but the scientific work of Rosalind Franklin was not recognized. The picture on the right pannel illustrates the schematic respresentation of the double-helix model of DNA proposed by Watson and Crick and published in the prestigious Nature journal (view the manuscript here).

In 1990 the second major milestone in the genetics of the XX century started with the sequentiation of the human genome in the "Human Genome Project", that concluded in 2003. A working draft of the Human Genome was completed and simultaneously published in Nature and Science on February 15, 2001.

New challenge in molecular biology: the ENCODE project

ENCODE (acronym of ENCyclopedia Of DNA Elemens) is a project funded by the National Human Genome Research Institute to identify all regions of transcription, transcription factor association, chromatin structure and histone modification in the human genome sequence. Thanks to the identification of these elements, 80% of the components of the human genome now have at least one biochemical function associated with them, in particular, the non-encoding regions. In this link to the ENCODE project, the most relevant results obtained in relation to the organization and regulation of the genes are shown.

The identification of new elements of the human genome has revealed new insights into biomedical research, as you can see in this video:

The central dogma of molecular biology consists of three main processes: DNA replication, RNA transcription and protein translation. Some of the basic techniques used in biomedical research are shown below:

Molecular techniques for the study of DNA:

• Analysis of numeric chromosomal anomalies: cariotype.
• Analysis of structural chromosomal anomalies: FISH 
• Detection of mutations: RFLP.
• Study of gene function: transgenic animals.

Molecular techniques for the study of RNA:

• Conversion of RNA to cDNA: reverse transcription.
• Gene expression analysis: conventional PCR, real-time PCR and microarrays.

Molecular techniques for the study of proteins:

• Quantification of proteins: ELISA, western-blot and proteomics.
• Localization of proteins: immunohistochemistry.
•  Study of protein function: stimulation of cell cultures, inoculation in experimental animals or patients.