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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.