Efficient , high-volume iPSC manufacturing opens new doors Lab workers at universities, life science institutes, consumer health and pharmaceutical companies use Human Induced Pluripotent Stem Cells (HiPSC) for basic cell biology research and genetic disease modeling, as well as for drug screening, development, efficacy and toxicity assessment. Being donor/patient-specific, HiPSC open tremendous possibilities for a wide variety of personalized studies in biomedical research. The process starts with adult skin cells (human dermal fibroblasts)
Efficient , high-volume iPSC manufacturing opens new doors
Lab workers at universities, life science institutes, consumer health and pharmaceutical companies use Human Induced Pluripotent Stem Cells (HiPSC) for basic cell biology research and genetic disease modeling, as well as for drug screening, development, efficacy and toxicity assessment. Being donor/patient-specific, HiPSC open tremendous possibilities for a wide variety of personalized studies in biomedical research. The process starts with adult skin cells (human dermal fibroblasts), which are re-programmed (induced) back into a primitive (pluripotent) state, yielding cells that can differentiate into multiple distinct lineages.
Despite the vast potential of iPSC, scientists using the technology voice a common theme: "We wish we could spend more time generating publication-quality data, and less time, money and resources reprogramming, growing and differentiating iPSC." In fact, lab heads and their staff report these laborious iPSC-related tasks can demand up 60-75% of their cell culture time, compared to productive stretches probing meaningful scientific questions. In short, the manual process of expanding stem cells can be labor intensive and time consuming.
But what if large numbers of iPSC could be made effectively, efficiently and under tight quality control? Let's say a billion high-quality, consistent iPSC from a single donor could be reprogrammed and then expanded in about a week - Would that get your attention? Such achievements are happening now, made possible through high-volume manufacturing that produces affordable, reliable human iPSC for life science discovery. In June, 2016 a partnership was established to revolutionize cell-based research, accelerate the understanding of disease and facilitate the identification of new therapeutic compounds. Cell Applications, Inc., a primary cell industry leader headed by CEO & co-founder James Yu, and StemoniX, an iPSC innovator led by CEO Ping Yeh, aim to bring researchers access to high-volume, cost-effective human iPSC.
A press release announcing the CAI-StemoniX partnership describes how high-volume biomanufacturing will change the paradigm by saving labs critical time, budget allocation and personnel resources. As stated in the release, the advance "will enable more research labs, regardless of size, to afford the large cell amounts and consistent quality necessary to advance scientific research." The joining of human iPSC generation and differentiation technologies, together with automated manufacturing, means iPSC and derived lineages can now be produced at an affordable, virtually unlimited scale, for even the largest research and screening projects.
The June announcement coincided with CAI's initial launch of these mass-produced HiPSC to the research community, from standard cryovials containing one million cells, to custom-generated lots containing hundreds of millions, or billions of cells. These integration-free HiPSC are generated with the RNA-based Sendai virus to deliver reprogramming factors to donor skin fibroblasts. Since the virus does not go through a DNA phase, its genetic material and transgenes do not integrate into the host cell genome. As for QC, the cells are validated for viability, karyotype, pluripotency, plating efficiency, morphology, passage number and lack of contamination. The cells display classic pluripotent stem cell markers and morphology, with a high nucleus to cytoplasm size ratio. Conforming to the demands of many applications, the cells are amenable to cultivation in serum-free media, independent of feeder cells and feeder-conditioned media.
Now Let's Talk iPSC Differentiation
Human iPSC have the potential to differentiate into multiple cell derivatives (Image StemoniX)
Ectoderm (Neuronal marker beta III tubulin / TUJ1) Mesoderm (Smooth Muscle Actin / SMA) Endoderm (Hepatocyte Nuclear Factor 3 Beta / HNF3b)
Examples of iPSC-Derived Lineages
Human iPSC-Derived Neural Stem Cells (i-HNSC)
i-HNSC are a homogeneous, self-renewing and multipotent population derived from Human Induced Pluripotent Stem Cells (HiPSC). The Image at right shows i-HNSC express characteristic markers such as the neural stem cell-specific intermediate filament protein Nestin (green) and the neural stem cell-specific transcription factor SOX1 (red). Nuclear staining with the DNA-binding stain DAPI (blue) is included as a reference. (Image StemoniX)
Under controlled conditions, i-HNSC possess the ability to consistently generate high yields of functional neural cells like neurons and glia. i-HNSC provide researchers a unique model to study neural development, neurotoxicity, differentiation, electrophysiology and disease modeling. Functionally mature human iPSC-derived neurons are essential to development of cell-based models with increased physiological relevance. Human iPSC-derived neurons and astrocytes, when co-cultured in vitro, enhance neuron phenotypic maturation and long-term survival. When cultured in defined differentiation media, i-HNSCs naturally generate a balanced population of neurons and astrocytes, providing a unique model that more closely resembles normal human brain physiology.
Image: Differentiation potential of iPSC-Derived Human Neural Stem Cells. In optimal conditions, i-HNSC differentiate into Neurons, as indicated by immunofluorescent staining for Microtubule-associated protein 2/MAP2 (green), which is essential to neuritogenesis. They can also differentiate into Astrocytes stained for glial fibrillary acidic protein (GFAP, red). Nuclear staining with DAPI is included for reference (Image StemoniX). The merging of these three images is captured in the bottom right panel.
Neuronal Multi-electrode arrays (MEA) measure real-time network activity of cultured human neurons. Likewise, MAE measurements in i-HNC-derived neurons show the presence of a mature, synchronized neuronal network with optimal electrophysiological activity, which can be modulated by neurotransmitters or small compounds.
Multi-electrode array of i-HNSC-derived Neurons (StemoniX)
Human iPSC-Derived Cardiomyoyctes (i-HCm)
As cardiac muscle cells, i-HCm make a powerful in vitro platform to study heart physiology & disease, and to assess therapeutic compound efficacy, safety and toxicity. Highly specialized, sensitive cardiomyocytes contain contractile units called myofibrils, and large amounts of mitochondria, which produce energy in the form of ATP, making them highly resistant to fatigue. Within the first quarter of 2017, CAI plans to introduce i-HCM in conjunction with StemoniX for life science research. Fully functional cells have already been successfully made and tested, and CAI will leverage this pre-launch period to engage in dialog with scientists to best understand their R&D needs and cell utilization patterns. Each lot of these i-HCm are derived from the iPSC of a single human donor, express typical cardiomyocyte markers, and demonstrate strong post-thaw viability and plating efficiency.
Image: Human iPSC-Derived Cardiomyocytes. Cryopreserved i-HCm 7 days after thawing & plating, immunofluorescently stained for the thin filament regulatory protein Cardiac Troponin T (cTNT, left, green) a heart-specific tropomyosin-binding subunit of troponin, the regulatory complex responsible for the calcium sensitivity important in proper muscle contraction, function and myofibril formation. Cells in the middle panel are stained for Sarcomeric alpha Actinin (S?Act, red), a cross-linking protein that anchors actin to intracellular structures in cardiac muscle. DNA in cell nuclei are counterstained with DAPI (blue).
Applications of i-HCM include:
Cardiac safety and toxicity: Cardiotoxicity has been known to halt late stage drug development or even spur the removal of previously approved drugs due to safety concerns. Through the CiPA initiative, which brings together researchers from institutions such as the FDA, academic institutions and pharmaceutical companies, i-HCm are being evaluated as an integral tool for the safety assessment of developing drugs. Cardiac Disease modeling: To date, interesting phenotypes of several genetic-based cardiomyopathies have been successfully replicated in a dish through the use of patient-specific i-HCm. Examples include Familial Hypertrophic, Dilated, and Arrhythmogenic Right Ventricular Cardiomyopathies. Independent assays: From electrophysiology and multi-electrode array analysis to high content microscopy and viability screens. High numbers of i-HCm are generated using the robust StemoniX manufacturing protocol. Their chemically-defined culture conditions are serum-free, feeder-free, integration-free and do not purify the cells through genetic selection, reducing the risk of genotoxic stress due to molecular manipulation.
Importantly, these i-HCm preparations include other relevant cell types present in the human heart, thereby increasing physiologic relevance, cell maturity, health and function, and more closely reflecting cell populations in a healthy cardiac tissue. Humans aren?t walking around with hearts made of 100% cardiomyocytes, and many argue neither should i-HCm preparations reflect such an artificial composition. Instead, the vials contain at least 40% i-HCm, as well as other naturally occurring cardiac cells, namely fibroblasts,endothelial cells and smooth muscle cells. The normal cellular composition and cell-cell interactions may help reduce experimental artifacts, false positives or negatives that could result from preparations enriched only for cardiomyocytes.
Cardiomyocytes derived from Human Induced Pluripotent Stem Cells (iPSC) spontaneously pulsate (beat) in vitro. This wave propagation indicates that iPSC, induced from skin cells (Human Dermal Fibroblasts), can then be coaxed into a functional heart cell lineage.
Heart Cells derived from iPSC beat in Culture Dish (Cell Applications, Inc.)
Researchers at the Cell Applications, Inc. Center for Primary Cell Technology & Innovation have generated mini heart tissue that pulsates (beats) outside the body. The achievement combines two of today's most groundbreaking, innovative technologies, namely iPSC and 3D Cellular Bioprinting. In this particular case, CAI reports successful 3D Bioprinting of iPSC-derived cardiomyocytes. A Cyfuse Regenova Bio 3D Printer fabricated the iPSC-derived cardiomyocytes into tissue spheroids. Cyfuse provides a three-dimensional organ regeneration platform using their novel robotic system. The Regenova facilitates fabrication of 3D cellular structures by placing cellular spheroids in fine needle arrays via the ?Kenzan method?, according to pre-designed 3D data. The spheroids then fuse to other spheroids, which can in turn further combine to create a variety of tissue shapes.
Stem Cell-Derived Heart "Organoid" Beats (Cell Applications, Inc.)
To begin the iPSC conversation and engage with technical experts, contact Cell Applicationstoday.
Aortic Endothelial Cells and Smooth Muscle Cells uncover mechanisms behind Atherosclerosis, Inflammation, Vascular Disease and Cardioprotection Intimal hyperplasia, a vessel thickening seen in atherosclerosis, is common after vascular injury, angioplasty or bypass surgery. Such insults trigger proliferation and migration of vascular smooth muscle cells, creating a lesion. Studies suggest testosterone protects men from atherosclerotic cardiovascular disease and plaque formation. This sex steroid mainly acts via the androgen receptor (AR), exp
Aortic Endothelial Cells and Smooth Muscle Cells uncover mechanisms behind Atherosclerosis, Inflammation, Vascular Disease and Cardioprotection
Intimal hyperplasia, a vessel thickening seen in atherosclerosis, is common after vascular injury, angioplasty or bypass surgery. Such insults trigger proliferation and migration of vascular smooth muscle cells, creating a lesion. Studies suggest testosterone protects men from atherosclerotic cardiovascular disease and plaque formation. This sex steroid mainly acts via the androgen receptor (AR), expressed in endothelial & vascular smooth muscle cells. Wilhelmson and co-workers from the University of Gothenburg recently offered new insights on the topic. Published in the journal Endocrinology (2016), they show testosterone inhibits migration and proliferation of Human Aortic Smooth Muscle Cells (HAOSMC). The in vitro data supports in vivo observations, suggesting deficiency of the testosterone receptor AR may increase intimal hyperplasia. Testosterone supplementation, which continues to increase in elderly men, appears to offer some levels of cardiovascular protection. This study supports the contention that additional mechanisms, such as direct effects on the vascular wall by androgens, could play important roles in cardioprotection. Future research may uncover potential therapeutic roles for selective androgen modulators.
During extravasation, circulating granulocytes reach sites of infection and inflammation. Pathogens trigger expression of adhesion molecules, initiating the rolling and adhesion of neutrophils to the vascular endothelial lining. One of these adhesion molecules, the glycoprotein P-selectin (SELP), transports to the endothelial cell surface. There, SELP mediates rolling by binding to its ligand on the neutrophil surface. In a recent study from Research in Veterinary Science, Chen and colleagues find that activation of Bovine Aortic Endothelial Cells (BAOEC) enhances SELP-mediated leukocyte attachment. The study provides novel evidence that high SELP polymorphism rates may potentially influence not only leukocyte migration, but also fertility. Both processes are key to successful performance in dairy breeds. Some aspects from this study in bovines could translate to humans, where P Selectin gene polymorphisms are associated with pregnancy loss. Further work may deduce whether such gene changes influence immunity, fertility, embryo and placental development.
The protein Adiponectin, produced by perivascular adipose tissue and detected in plasma, helps thwart vascular disease. Adinopectin improves insulin sensitivity and coronary blood flow, and safeguards against endothelial dysfunction, inflammation and glucose-induced oxidative stress. It provides these cardioprotective properties by elevating production of calcium and nitric oxide, the latter through activation of the endothelial NO synthase signaling pathway. The obese, type 2 diabetics and those resistant to insulin show decreased levels of adinopectin. InLife Sciences, Grossini et al now show adiponectin treatment induces Porcine Aortic Endothelial Cells (PAOEC) to increase NO release and calcium movements during endothelial dysfunction like that caused by high-glucose. This signaling involves Akt, ERK1/2 and p38MAPK downstream AdipoR1. The results add new information about the control of endothelial function elicited by adiponectin in both physiological and pathological conditions. From these data, one can envision a beneficial role for adinopectin in endothelial function, and in preventing glucose-induced endothelial damage.
For decades, immortalized cell lines have performed admirably in cell-based assays, biomanufacturing, and basic scientific work. ?Let?s give them due credit,? says Daniel Schroen, PhD, a vice president at Cell Applications (San Diego, CA). ?They are a mainstay.? Primary cells have similarly been part of life sciences research, and demand for these cells is at an all-time high. Compared with immortalized cells, the key advantages of primary cells are their functional and genetic fidelity. ?The information derived from primary cells is clos
For decades, immortalized cell lines have performed admirably in cell-based assays, biomanufacturing, and basic scientific work. ?Let?s give them due credit,? says Daniel Schroen, PhD, a vice president at Cell Applications (San Diego, CA). ?They are a mainstay.?
Primary cells have similarly been part of life sciences research, and demand for these cells is at an all-time high. Compared with immortalized cells, the key advantages of primary cells are their functional and genetic fidelity.
?The information derived from primary cells is closer to physiologic relevance because these cells lack the genetic changes that allow indefinite in vitro cultivation,? Schroen adds.
Primary cells present challenges, however. Their supply is limited, they are difficult to acquire and isolate, they are intolerant to all but very narrow culture conditions, and they don?t last long: Primary cells offer at most 15 to 20 passages, or doublings, before they die out, whereas immortalized cell lines go on forever. However, these issues are usually manageable with careful optimization of isolation and culture methods, media, and nutrient feeds.
Specialized function, specialized conditions
Because primary cells are highly specialized, they require individualized environmental and nutritive conditions for optimal growth and maintenance of the desired phenotype. This is typically achieved through application of specific growth media, supplements, and extracellular matrices or other application- specific conditions. ?The goal is to preserve the cells? original functionality,? says Robert Newman, PhD, director of R&D for ATCC Cell Systems (Manassas, VA).
In carrying out their specialized activities, primary cells have distinctive receptors, enzymes, and signaling pathways that work through unique triggering mechanisms that are absent in non-primary cells. Thus nearly every vendor of primary cells also offers precisely defined media formulations for specific cell lines, e.g., fibroblasts, or kidney or endothelial cells. As with immortalized cells used in biomanufacturing, where the goal is achieving higher protein titers, media formulations for primary cells are optimized by component to achieve in vitro fidelity to their in vivo phenotype.
The finite life of primary cultures recapitulates what occurs in vivo, where cells differentiate, take on their specialized functions, and stop reproducing. Senescence is a complicated process. ?Certain signaling pathways cause terminally differentiated cells to stop replicating in the body,? Newman says. ?When you put those cells into culture, they follow a similar trajectory toward senescence. That?s typical for differentiated cells but not for primitive cells, which are at the opposite end of the spectrum with respect to proliferation.?
Some primary cells, such as terminally differentiated hepatocytes and neurons, are post-mitotic, and under normal circumstances do not proliferate at all, either in vivo or in culture. While liver tissues repair themselves in vivo, these processes appear to be mediated by resident stem cells and complex triggering signals. Cultured primary hepatocytes barely last for one week without highly specialized media formulations.
Pharmaceutical companies increasingly rely on primary cells throughout research and development. Some isolate and culture the cells on their own, but many turn to companies like Cell Applications for fully authenticated, ready-to-use cells. Cell line authentication has become a huge issue with journals and funding agencies, and companies with isolation expertise usually lack the tools to prove their cells are what they claim them to be.
Pharmaceutical drug discovery has become a key testing ground for many advances in the life sciences, and primary cells are no exception. Primary cells are used during very early compound screening through initial low-throughput, low-content assays. ?This is when companies are looking for hits that are physiologically relevant,? Schroen explains. Primary cells may be used alone or to complement testing in immortalized cells. Later, as screening activity picks up and for practical reasons, companies turn to immortalized cells that respond similarly to primary cells, usually through high-throughput screening. After this phase, they often return to primary cells to fine-tune compound evaluation or to evaluate in vitro efficacy and toxicology.
At this stage companies are particularly interested in three-dimensional primary cell constructs formed either through bio-printing or by physically layering cells onto a suitable matrix. Cell Applications is one of several companies that offer such 3-D cell models (for a more complete treatment of 3-D cell culture see Lab Manager, September 2015). Due to their structure and more natural cell-cell interactions, 3-D cultures often supplement 2-D or suspension cultures with additional physiological relevance and data. They also significantly reduce the costs, lengthy time requirements, and potential ethical concerns of animal testing.
Cell Applications has developed a layered 3-D human cell model for skin, where human epidermal keratinocytes differentiate into a stratified squamous epithelium-like construct. Their 3-D airway model consists of human bronchial epithelial cells that form a pseudostratified epithelium. The former is of great interest to pharmaceutical and cosmetics companies, offering an in vitro system for studying wound healing, UV damage, and the absorption, penetration, metabolism, and toxicology of topical agents. The company?s bronchial cell products are used to study potential treatments for airway infection, lung injury, mechanical and oxidative stress, inflammation, pulmonary diseases, and smoking.
Because of the complexity of a typical organ, its need for blood vessels, and the sheer number and diversity of functional cell types, Schroen believes it will be a challenge for 3-D bio-printing companies to develop large, fully functional organs like a kidney or liver. However, he does see the benefits of using what amount to smaller, printed tissue components for pharmaceutical drug and cytotoxicity screening. The foreseeable future could also move three-dimensional cell printing into the clinical realm, for instance with tissues to repair blood vessels or skin, or someday even replace diseased structures within major organs.
Acquiring Primary Cells
?There?s a significant learning curve involved in acquiring primary cells, and the process is time- and resource-intensive,? Schroen tells Lab Manager. Each major primary cell type requires unique isolation and purification protocols. Moreover, somatic primary cell lines show varying susceptibilities to their physical and chemical environments, to enzymes, mechanical disruption, or agitation. By contrast, immortalized cells are relatively hardy, typically thriving in standard off-the-shelf media.
Before releasing a new cell, Cell Applications undertakes an optimization process to assess which media components produce the most desirable cell performance, which, depending on the cell type, can include cell viability, morphology, population doublings, cell surface marker expression, or cell signaling cascades. Some cells will not grow outside of serum-based media, while others do much better in animal- component-free or chemically defined media. Human primary cells have traditionally been obtained in collaboration with research hospitals or tissue banks from surgical excisions, and from post-mortem organ and tissue donations. Nonhuman tissues are sourced primarily from in-house animal facilities, companies that raise animals specifically for research purposes, or carefully monitored animal processing facilities. All tissues, whether animal or human, carry detailed donor information such as age and sex, while the human donor profiles also include ethnicity, any diseases or pathologies, medications taken, and cause of death. It?s important with primary cells to have available different lots, for example certain ethnicities, tissues, age, and disease status. ?If we get a very narrow request, we can tap into our resources to obtain those cells,? Schroen says. ?Some pharmaceutical companies request one donor but four or five different tissues; others request a single cell type from multiple donors of different backgrounds.?
Stem Cells: Not Quite There Yet
The potential of stem cells to retain multi-lineage potential and proliferate extensively in vitro provides new avenues for cell-based therapy in the restoration of damaged or diseased tissue. Stem cells, particularly induced pluripotent stem cells (iPSCs), are also a potential source of primary cells. Cell Applications has a special interest in iPSCs through their work with iPSC discoverer Shinya Yamanaka, who won a 2012 Nobel Prize for his work. In this case, CAI supplied Dr. Yamanaka?s team with human dermal fibroblasts (skin cells), which they coaxed back into a pluripotent state, forming a potential source of primary human cells of nearly unlimited number and cell type. Bone marrow stromal cells, also known as mesenchymal stem cells, can produce bone, cartilage, and fat cells, as well as neuronal and hepatocyte lineages. Multipotent neural stem cells differentiate into neurons, astrocytes, and oligodendrocytes that make up the central nervous system.
This raises the question of the potential for using stem cells, in particular iPSCs, as starting points for generating primary cells. Compared with tissue harvesting, iPSCs have the advantage of a nearly endless supply. They may be expanded to large numbers and stored cryogenically for future expansion or differentiation. ?You can control for donor-to-donor genetic variability within a large set of experiments if you can generate larger banks of cells from the same source,? Newman explains.
But iPSC-derived primary cells are not quite identical to mature cells harvested from living tissue. Harvested cells tend to retain their native phenotype in culture, albeit for short periods of time, and behave similarly to cells in vivo. Cell-based drug or toxicology assays based on them more closely represent what occurs in intact organisms. ?They already possess their fully functional activity,? Newman adds. Since iPSCs were first reported in 2006, biologists have identified conditions, including growth factors and media, that cause iPSCs to differentiate into dozens of cell types. Performance of these cells in assays has been mixed, however, because the cells, while resembling skin, kidney, or heart cells in overt aspects, often do not replicate the behavior of fully mature, differentiated cells.
?Equivalence of iPSC-derived cells to mature in vivo cells varies,? Newman explains. ?iPSC-derived terminally differentiated cells do not capture the entire mature phenotype of primary cells. You can make hepatocyte- like cells, or cardiomyocyte-type cells, but they usually exhibit functional features of less mature cells.?
Interestingly, the success of iPSCs in preclinical regenerative medicine studies results from their achieving fully differentiated status when introduced to their familiar biological niche. ?We haven?t yet identified all the factors that trigger full differentiation in vitro,? Newman notes. ?I?m confident that future advances in substrates and media will help solve this problem, and create powerful, robust in vitro assay tools.?
By Angelo DePalma | February 09, 2016 | Lab Manager