Creating transplantable human eyes in a petri dish
Creating transplantable human eyes in a petri dish: A quest that Dr. Sangeetha Kandoi is determined to tread on
Ever since I worked on the organoids story for BioSpectrum India magazine, I wanted to sit down with my dearest friend, Dr. Sangeetha Kandoi , who has done marvellous work in stem cell research. Dr. Kandoi’s research on retinal organoids has earned her accolades and appreciation, and she remains motivated to further her research in organoids.
This detailed Q&A with Dr. Kandoi reveals how 3D organoids are revolutionizing stem cell research and opening numerous possibilities. With the advent of iPSC, relying on animal models for drug testing is now passé, says Dr. Kandoi.
Read on to gather first-hand knowledge on 3D organoids and their benefits.
The potential of stem cells is ‘HUGE’ and needs to be ‘TAPPED’
From your perspective, what are the most significant advancements in organoid technology in the past few years that hold the most promise for drug discovery and development?
3D organoids have emerged as a valuable tool for modelling any human ‘disease-in-a-dish'. Developing a complex tissue (and not organ) vis-à-vis the state-of-the-art, 3D organoids precisely recapitulate the paradigm of in vivo host tissues in contexts to developmental timelines and cell types. Organoids (derived from stem cells) have transformed clinical care with guided personalized medicine.
The potential of stem cells is ‘HUGE’ and needs to be ‘TAPPED’! I recollect embarking on my career as a stem cell biologist under the mentorship of Dr. Ajit Kumar, PhD (Retired Chief scientific officer-R&D Unit, LifeCell International Pvt Ltd, Chennai) back in 2005. There were no smidgeons of induced pluripotent stem cells (iPSCs) in those days, and we worked with ‘multipotent’ adult stem cells.
Then, the breakthrough of ‘pluripotent’-iPSCs by the Nobel Laureate, Shinya Yamanaka [in 2006 (from mice) and 2007 (from humans)] revolutionized the field of regenerative biology and medicine. The ability to reprogram the somatic cells (For example, skin fibroblasts, urine cells, mononuclear cells from peripheral blood, neural progenitor cells, etc.) into embryonic-like cells (iPSCs) was ascertained by simply expressing ‘four magical transcription factors’ encoding Oct4, Sox2, Klf4, and c-myc. Expression and maintenance of these ‘niche factors’ permits the iPSCs to remain ‘undifferentiated’, while enabling them to ‘differentiate’ into any required cell types for in vitro studies including drug screening and validation at ease.
All these remarkable discoveries of science intensified my passion to pursue my graduate training in the Stem cell and Molecular biology laboratory of Dr. Rama Shanker Verma, PhD, Professor, IIT Madras (2013-2018). During my graduate training, I had an opportunity to move to United States for 2-years (2015-2017), wherein I was able to successfully model a human disease-in-a-dish via 2D culture by collecting samples from South Asians individuals living in the United States with Hypertrophic cardiomyopathy.
By collecting just a few drops of their blood/urine from patients with HCM, I followed Dr. Yamanaka’s magical recipe to make iPSCs which were further utilized to differentiate into unipotent-contracting (beating) cardiomyocytes in a dish. From feeling a heartbeat (by placing my hand on the chest) as a kid to watching cells beating on one bright day (after countless trial-and-error experiments) was truthfully an awe-inspiring experience for me. Watching the beating cells-in-a-dish was an ‘eye candy’ experience, and I just could not take my eyes off from the microscope for many many days after that. This was one of the most and first fascinating scenes of my scientific career (PMID: 29641836).
While I was still celebrating this little joy of admirable science, researchers across the globe were actively working on advancing the technology in transforming from 2D to 3D differentiation for precise understanding of the disease etiopathology, drug discovery and regenerative medicine. The beginning of 3D-organoids permitted to construct the native (or in vivo) tissue in a dish displaying heterogeneous cell types organized in a complex architecture. Since most tissues in our body exists in 3D fashion, ‘mimicking’ them in the form of 3D-organoids implied them to be a remarkable tool for in-depth biological studies. All these technological advancements compelled me to shift gears to venture into this latest era of 3D-organoid research, more specifically into Retinal Organoids (PMID: 37440085).
Fortunately, in August 2018, I had the honour of working with Dr. Jacque L. Duncan, MD, Chair & Retina Specialist in the Department of Ophthalmology at the University of California San Francisco, CA, USA. Dr. Duncan presented me one of her best-loved projects, where one of her patients had been progressively losing his vision due to Retinitis Pigmentosa (RP), a most common group of inherited retinal degenerations (IRD). More than 250 mutations had been linked to be a causative factor of RP, but this specific patient was unique and was known to harbour multiple copies of rhodopsin (RHO) [First identified and described by Dr. Duncan at the IOVS conference proceedings, 2019).
Due to my prior experience of modelling a disease using patients’ stem cells, I grabbed this assignment assertively. Developing a 3D retinal organoid model (Mini Retina) was new to me but with substantial hard work and a determination to thrive, I successfully built a ‘mini retina’-in-a-dish. I want to let the readers know that building a retina is ~a-year long laborious and committed process of both the cells and my involvement in nurturing and grooming them. This has been one of the longest experiments I had ever done in my life, where I couldn’t apply any ‘brake’ or take a weekend off. Fortunately, in a year’s time, these mini retinas which I generated were histologically indistinguishable to in vivo retina displaying all the seven retinal cell types. But interestingly, the differentiated 3D-mini retinas from the patient displayed a pattern of ‘mis-localized excessive Rho protein’ in the ‘cell body’ as opposed to the ‘outer segments’ of the light-sensing cells (aka., Rod photoreceptors). This result was bizarre.
Understanding the reason of vision loss in this patient was crucial and this was successfully accomplished by building the patient’s retina-in-a-dish. The immediate next burning desire was how could we fix this issue as these mini retinas (although ‘diseased’) became my dear babies in a year’s time by closely taking care of them every day. And the goal was to integrate the knowledge of ‘bench research’ findings to ‘bedside’ clinical care for patients experiencing with vision loss. So, then the ‘mini retinas’ of the patients made in the lab were utilized as a most powerful tool for drug testing. A drug (Photoregulin 3; PR3) was tested on this patient-specific retinal organoids (~300-days-old). In a week’s time of exposure to PR3, trafficking of Rho protein from the cell body to inner/outer segments of Rod photoreceptors was visualized. The establishment of this proof-of-concept implicate 3D-retinas as a valuable preclinical model for attenuating the catastrophic vision loss in this patient (PMID: 38661530).
Currently, there are numerous drugs available at the pharmacy counter/prescribed from doctor’s office to cope up with our day-to-day common problems like fever, headache, diarrhoea, etc. Preliminary results from my retinal organoid study with PR3 drug offers a promising hope to provide a quality life for people suffering from blinding diseases. Few years from now, it would be realistic to see patients having accessibility to drugs like PR3 at the pharmacy counter for fixing their progressive vision loss.
Few years from now, it would be realistic to see patients having accessibility to drugs like PR3 at the pharmacy counter for fixing their progressive vision loss.
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How could these research advancements potentially address current limitations in traditional drug testing methods - with specific reference to animal testing models?
iPSCs are ‘powerful’ and ‘flexible’ friends. This means they are potentially ‘immortal’ and have ‘multi-lineage’ differentiation potential making them as an unlimited source of tissue regenerations. Prior to the discovery of iPSC, researchers had to rely on cadaver tissues to obtain the terminally differentiated ‘unipotent’ cells. Nonetheless, with the ability of iPSCs to be able to differentiate into all the 220 types present in the human adult body as like embryonic stem cells without any ethical issues is a boon for biomedical research and to advance the field of therapy.
The historically used preclinical model systems including the 2D cell lines and US FDA mandating traditional animal models were marked as ‘gold standard’ to earmark the testing of any new drug prior to entering human clinical trials. However, it is time to rethink numerous challenges, limitations and intrinsic flaws questioning the scientific merit of such models to uncover and bridge the gap between the mouse and humans.
The most essential challenges include the failure to mimic the physiological conditions and the biological variation, i.e., the xenograft model (species differences). Undoubtedly, mice have been a driving force and a valuable model to lay down the foundation for approximating the understanding of human health and diseases. Nonetheless, ‘Mice are not humans’, as they differ from humans in numerous aspects - ~3000 times larger in size implicating poorly mimicking physiology functions, 97.5% (and not 100%) comparable genomic similarity, decreased metabolic rate in comparison to mice, trinocular vision in humans as opposed to binocular vision in mice, diurnal habitat in humans contrasting to nocturnal mice, longer life span with ~70 years in humans to just ~2 years in mice and many more!
A complementary and a clear alternative with the advancement of organoid technology have been identified to circumvent the required challenge for the need of the human model system for research and clinical settings. Most often the drug tested on traditional animal models such as mice and rats has led to notable (i) unfavorable adverse effects in humans (ii) deleterious delay in pre-clinical trials (iii) high toxicity-related failure beyond pre-clinical and human clinical trials (iv) significant time and $$, and (v) retraction of many drugs from the market annually accompanied by substantial financial forfeiture and socio-economic burden. For instance, the commonly prescribed Ibuprofen drugs are utilized for relieving pain, fever, and inflammation in humans, while the same have been proven to be toxic in rats.
In this scenario, the preclinical pharmaceutical tested data obtained from the innovative 3D-organoids will potentially be robust and booming, than the failure rates in animals. Human 3D organoid model system serve as a fundamental contributor in the success of a translational therapeutics by intersecting the ‘bench’ findings-to-‘bedside’ clinical care. The scientific validity of drugs on human model system will rigorously weed out drugs posing significant adverse human effects and will benefit millions of patients worldwide with advanced clinical care. The convergence of organoid technology for pre-clinical trials will undoubtedly delineate the future challenges of rigor, reproducibility, safety, and efficacy of drug responses, benefiting the patients with successful clinical outcome along with animal welfare.
If in the future organoid-based drug testing would replace or significantly reduce the reliance on animal models, what steps would be necessary to achieve this, and what are the potential benefits and challenges associated with such a shift?
Along with animal models, cell lines (2D cultures) have been an ardent model in the development of drug testing owing to its relatively simple inexpensive and easy to expand features. However, two main limitations including (i) representation of single cell type and (ii) the failure to reflect the closer biological communication from the adjacent cells as in in vivo have often limited their use and predictability in experimental research.
The evolution of 3D-organoid cultures has been the guardian under these scenarios broadening their prospect in various fields of medical research. Although, 3D-organoids are still at the infant stage of research progressive development has been made over the last decade since its preliminary description by Sato et al in 2009.
Several researchers including me have been captivated by 3D- retinal organoid technology as they are composed of self-assembled, differentiated cells and are analogous to the human in vivo retinal tissue. The future era relies on personalized and accurate treatments unlike the application of single drug for all patients. Consequently, a clear milestone is being laid down to utilize 3D-organoids as a mainstream viable alternative to the existing animal models.
The first and the foremost requirements is to establish a biorepository of stable and renewable source of fully characterized patient-specific iPSCs with explicit consent from humans of diverse ethnicity and genericity for numerous diseases, and with varying disease severity. The availability of such repositories can aid to initiate the research studies and will expand the horizon of modelling disease to developing therapies via 3D organoid technology.
Conspicuously, the 3D organoid differentiation demands valuable resources including (i) skilled labor for generating tissue-specific organoids (ii) prerequisite time to establish matured cell types for uncovering the pathological consequences of genotype-phenotype correlation (iii) long-term continuance need of aseptic conditions for viable maintenance of organoids (iv) multi-step protocol to lead the way of differentiation trajectory making them cost-intensive. Establishment of organoid biobanks with several sample sizes retain the individuals original genetic and epigenetic state which are crucial to envisage the drug responses with diverse ethnic backgrounds.
Herein, the in vitro 3D organoids bring a hope for safety, long-term stability, and functionality with the tested drug over time. Numerous studies including one of my own published research projects have augmented our understanding to predict a patient’s response by testing a drug on patient-specific 3D-organoids. Despite the above forementioned advantages, organoids have certain significant limitations in dispensing the message for precise therapeutic benefits. The absence of vascularity and immunological cues, primarily from the immune cells and factors describes them to lack the dynamic microenvironment as in in vivo tissue. Ongoing continuing efforts to overcome these shortcomings will break through the constraints with the goal to enhance the precision medicine in clinical settings.
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