INDIAN COUNCIL OF MEDICAL RESEARCH
Format for ICMR-ESSAY COMPETITION 2018
Title of the Essay:
Prediagnosis of diseases by measuring functional efficiency of intracellular organelles in organoids acquired from patient derived iPS stem cells
Essay:
Majority of existing diagnostic methods relay on detection of abnormal factors in the body fluids. Therefore diagnosis can be attained only after the manifestation of a physical symptom. Abnormalities in intracellular machinery precede the development of symptoms and infact it is the increased number of intracellular dysfunction that finally lead to the development of a gross anomaly. This in turn presents itself as a clinically relevant symptom. By culturing organoids in patient serum infused 3D matrix representing every organ of each individual, we aim to detect intracellular organelle dysfunction much early than the disease develops. Detection of mitochondrial depolarization in hepatocytes in alocholics. Thus our goal is to maintain a miniature of a human body.
Modern medical advances have helped millions of people all around the world to lead a much longer and healthier life. The innovations in the field of medicine have come so far, that we could even dare say that the balance on horrendous diseases like cancer seem to be slowly tipping in our favour, all of which we owe to the improvement in medical research over the years. Considering the current scenario where non infectious and endogenous diseases which develop based on a genetic predisposition and lifestyle habits are on the rise, there is a warrant for increased concern to attain an early diagnosis so as to prevent the disease altogether or start treatment as early as possible to give patients the best quality of life possible .
Imagine a situation where we could monitor patients 24/7 all around the year ,where we could diagnose and treat a developing disease much before it grows too rampant to be brought under control . Think of a time where the phrase “ We couldn’t catch it in time” would be used much less frequently in a doctor patient conversation .
Sounds too good to be true, does it not ? But I strongly believe that with use of the modern breakthroughs in molecular biology and through close collaboration of the field of medical research and clinical medicine; that this is possible .
The invention of differentiated cells which can be reprogrammed to become pluripotent which can in turn be transformed to other cell fates by treatment with defined factors has opened up new opportunities in clinical medicine. Induced pluripotent stem cells (iPSCs) has opened up unprecedented opportunities in the pharmaceutical industry, in the clinics and in laboratories. In particular, the medical applications of human iPSCs in disease modelling and stem cell therapy have been progressing rapidly. (Takahashi K & Yamanaka,2013)
Various researchers all over the world were able to use iPSCs to stimulate growth of various types of cells of the human body, from a single precursor colony by means of necessary stimulation. A testimony to this fact would be the preparation of pancreatic β-cells from human iPS cells with small molecules (Masaki Hosoya,2012) & the promise of iPSC-derived neurons (Dolmetsch & Geschwind, 2011).
The opportunities that iPSCs provide to the field of stem cell therapy are numerous . But the diagnostic significance of this innovation had been largely overlooked. The iPSCs generated from a patients sample is unique to him and can essentially act as a representative form of every major organ system of a patient when differentiated . This forms the foundation of the idea that I wish to present .
Screening for a disease on a cellular basis can alert us of the development of an anomaly in the human physiology much earlier than in the case of having to wait for it to develop into a grossly presentable symptom. For clarity on this aspect, let us consider an example of the case study “Severe Alcoholic Hepatitis in a Teetotaler” (Cyriac Abby Philips,2018). Here, a 44-year-old teetotaler male, pineapple farmer by occupation presented with classical clinical and biochemical features of severe alcoholic hepatitis associated with pruritus for 3 days . He denied over the counter medications and alcohol intake which were the most common etiological reasons his condition . For a while no explanation could be found so as to why the patient had developed alcoholic hepatitis. Finally the line of correlation was drawn to his chronic use of ‘dashamoolarishtam’, an age old Ayurvedic digestive drink which contained alcohol due to fermentation of various herbal extracts contained in it and to that of acute pesticide poisoning which he suffered due to exposure while spraying his pineapple farm. Biopsy of his liver revealed steatosis, which was a classic symptom of alcohol abuse. The lipid laid cells however would have manifested in the liver a much longer time before the onset of his symptoms that brought him to the clinic. Hence, in a situation where we might have been able to screen him on a molecular basis; his developing disease would have been curbed almost a year ago before it grew to cross a critical threshold and produce devastating effects on his body .
Donor cells representative of each individual is collected and differentiated to form representative colonies of each of his major organ systems. An average of 10 culture plates is maintained per patient . The cell cultures that would be running would include hepatocytes, pancreatic β-cells, neurons, bone marrow, cardiomyocytes etc. The first visit would involve a complete assessment including past personal and familial medical history as well as routine tests to assess his present health standard . Cells are collected for differentiation and 20 ml of blood is drawn from the patient .
The blood is centrifuged to obtain the serum. The cells after differentiation are maintained in a medium which has the patients serum in a standardised ratio so as to expose the differentiated cells to the cellular environment that is a perfect replica to that of the patients’. The experimental basis of this was derived from a simple experiment wherein two colonies of identical cell lines of the same age were grown in a medium that was mixed with serum of different age groups . The cells which were exposed to the serum of an older patient showed changes in its cellular morphology and growth pattern which were consistent with stress when compared to the other cell plate which received an adolescents serum which led us to believe that the factors present in the serum of an individual can help mimic the cellular environment inside the body for cultivating a tissue explant.(Jaivardhan et al. unpublished data)
The differentiated cells are then monitored through means of fluorescence microscopy, western blots etc in order to asses anomalies. Screening of the cells are done in an organised fashion based on the organelle structure considering the cellular morphology of each colony . Each anomaly in a particular cell line leads to a red flag that any one of a varied number of diseases might be evolving in the patient . A differential diagnosis of sorts.
Each deformity in the morphology of the numerous organelle in different types of cells indicate the development of an array of diseases. Therefore, the screening would be done on an organelle basis . If we were to consider the endoplasmic reticulum (ER) in pancreatic cells, an organelle that performs folding, modification, and trafficking of secretory and membrane proteins to the Golgi compartment, the maintenance of ER homeostasis in insulin-secreting β-cells is very important. When ER homeostasis is disrupted, the ER generates adaptive signaling pathways, called the unfolded protein response (UPR), to maintain homeostasis of this organelle. However, if homeostasis fails to be restored, the ER initiates death signaling pathways. New observations suggest that both chronic hyperglycemia and hyperlipidemia, known as important causative factors of type 2 diabetes (T2D), disrupt ER homeostasis to induce unresolvable UPR activation and β-cell death.(Back SH & Kaufman RJ, 2012) Therefore detection of ER stress in the β-cells do not point only to an increased chance of the patient developing type 2 diabetes, but it gives us an idea as to the fact that the person may be suffering from hyperglycaemia and hyperlipidemia .
In case of hepatocytes, the lipid laden micro vesicles cells can be easily demonstrated by use of BODIPY stains under confocal microscopy. Assessment of cardiomyocyte function can be based on the levels of exclusive translation factors such as GATA-4 and by observing if the cells are in any stage of hypertrophy. Hypertrophy beyond normal growth is the response of the myocyte by increased protein synthesis to various stimuli including hemodynamic, humoral and ischemic factors(Bisop Sp,1990). Hypertrophic cardiomyopathy (HCM) is the most common cause of sudden cardiac death in young individuals. In a study using induced pluripotent stem cell-derived cardiomyocytes (HCM-iPSC-CMs) from human patients, they found mitochondrial dysfunctions and ultrastructure defects by decreasing the stability of 16S rRNA, which led to reduced levels of mitochondrial proteins. The ATP/ADP ratio and mitochondrial membrane potential were also reduced, thereby elevating the intracellular Ca2+concentration, which was associated with numerous HCM-specific electrophysiological abnormalities. Therefore the mitochondrial polarity and character can also be assessed in cardiomyocytes to give early indications on the development of possible cardiomyopathies.
The development of neurodegenerative diseases can be monitored by various means such as assessment of autophagy in the iPSCs (autophagy linked degenerative diseases), chromosomal abnormality monitoring and the appearance of alpha-synucei in the neurons. Patient iPSC-Derived Neurons for Disease Modeling of Frontotemporal Dementia has already been carried out where they were able to isolate the deficient protein and link it to a mutation on chromosome number 3(Yu Zhang et.al,2017 March)(Despina Yancopoulou et.al, 2003). The appearance of Alpha-synuclei in Lewy bodies have been associated with the development of diseases such as Parkinson’s and can therefore give us an opportunity, if detected early, to help the patient lead a better quality of life. A vaccine approach targeting alpha-synuclein in Parkinson's patients is now in the clinic.As of January 2012, The Michael J. Fox Foundation had invested over $47 million in projects targeting alpha-synuclein. And if the development of the vaccine is successful, this method can act as a screening procedure to administer the vaccine to the affected .
A draw back of the idea is that it cannot be used to diagnose acute illness that the patient might suffer from or exogenous infections which may affect the patient . The ingenuity and instincts of the doctors are necessary to diagnose and treat patients in these scenarios . But in diseases which follow a chronic timeline which develops due to intrinsic factors, this idea can be potentially lifesaving and opens up a whole new realm of opportunities for medical researchers as well as doctors alike .
Maintaining 10 cell culture plates per person for a year would roughly need 10,000 rs, half of which would be used for culture media and other materials necessary for maintaining cell culture . If the project in its initial stages can be integrated with existent facilities carrying out studies in cellular biology, who would most probably have all the equipments needed for assessing the cels such as confocal microscopes,incubators,culture hoods etc ; then the collective cost per person would be less making the idea more feasible economically as well .
Although innovative breakthroughs are occurring in the field of medical research, its integration onto clinical practice is lagging to a great extent . The invention of induced pleuripotent stem cells (iPSCs) from precursor cells can essentially provide us with the leeway required to grow organoids corresponding to the major organ systems in a human body in cell culture plates. These cell colonies can act as representative samples of an individual when the iPSCs generated for each individual is grown from their own cell samples. When the cells are grown in a media mixed with the patients serum in a standardised ration, it would essentially act as a clone of the cells inside the patients body . Monitoring and assessment of these cells would allow us to identify and evaluate developing diseases based on disturbances in organelle morphology or function. Translation of this idea into clinical practice would help us diagnose the diseases in a timely fashion and perhaps even prevent or cure it completely. There wouldn’t be a necessity for the patients to develop clinically presentable symptoms, by which time it might already be too late or the disease might have done irreparable damage to the body of the patient . After all, I believe that no human being would disagree with the fact that timely prevention and palliative care would ease an patients life rather than delayed treatment, which may burden him financially or subject him to lead a debilitating life for the rest of his years
Applicant’s Full name- Jaivardhan A.Menon
Applicants College Name & address- Sree Narayana Institute of Medical Sciences
Applicant’s Contact no & Email ID: 8921194152, jaivardhanajakumarmenon@gmail.com
Name and Signature of College Principal/Director/Dean (with seal)