Introduction
Patient-derived induced pluripotent stem cells (iPSCs) offer exciting potential for disease modeling, regenerative medicine, and therapeutic development. In order to fully realize the potential of iPSCs, it is critical to develop culture and differentiation methodologies that are scalable and result in the most biologically relevant cell types.
What are naïve stem cells?
A pluripotent cell is one that has the capacity to develop into any cell type of an organism. However, there are two distinct states of pluripotency: naïve and primed. Naïve stem cells are derived from epiblast cells of the preimplantation embryo, while primed cells are derived from the postimplantation embryo. Although both naïve and primed stem cells are able to self-renew indefinitely, express OCT4, SOX2, and NANOG, and are able to differentiate down all three germlines, they are molecularly and functionally distinct.
A. X- chromosome activation in naïve cells - Cells grown in FGF (left) are showing X chromosome inactivation, where the cells grown in the naïve state using NME7AB have no X inactivation.
B. Naïve cells have a unique gene expression profile - RNA sequencing shows a completely different genetic fingerprint between cells grown in FGF and cells grown in the naïve state using NME7AB.
Despite the 10 years that have passed since the Nobel Prize winning discovery of iPSCs, their generation and expansion are still routinely done in the primed state using FGF-based media on feeder cells or extracellular matrix substrates. These methods are inefficient, labor-intensive, and relatively nonscalable. The use of naïve stem cells addresses these issues and provides several advantages over primed state cells, which are important for the future of stem cell therapies.
What is NME7AB?
Scientists have generated human stem cells that in some respects mimic mouse naïve cells, but their dependence on the addition of several extrinsic agents, and their propensity to develop abnormal karyotype calls into question their resemblance to a naturally occurring "naïve" state in humans.
Minerva Biotechnologies is the first company to generate human naïve state pluripotent stem cells using a single, naturally occurring human stem cell growth factor, NME7AB. This growth factor is expressed in the earliest days of embryogenesis, in the inner cell mass of human blastocysts, and is later turned off during development.
Minerva’s stem cell growth system has 3 components: 1) NME7AB as sole growth factor; 2) an antibody adhesion surface that captures MUC1* positive cells; and 3) a synthetic peptide having sequence of the MUC1* extracellular domain; this peptide acts as a ligand sink to break the pluripotency signal; it synchronizes differentiation and ensures that no pluripotent stem cells remain.
What are the advantages of naïve stem cells?
Improved iPSC Generation
Despite the desire to move away from the variability and labor associated with MEFs, scientists believe they need feeders in order to achieve high efficiency reprogramming. Generating naïve stem cells using NME7AB, instead of primed cells using FGF, provides high efficiency feeder-free reprogramming with improved cell line survival.
Scalable Cell Expansion
Large-scale production of human pluripotent stem cells has been one of the major challenges for translational applications of stem cell technology. In the naïve state, stem cell expansion is faster, scalable, and can be completely automated since it eliminates unwanted spontaneous differentiation, while maintaining a normal karyotype. The use of naïve stem cells provides a scalable, robust and economic method that yields over 1 billion high quality cells in 17 days (starting from just two 6-well plates).
Enhanced Directed Differentiation
Naïve stem cells do not have DNA methylation marks that commit the cells to certain developmental decisions. Because these earlier stem cells have a “clean slate,” they are more easily directed to develop into functional mature cells.
Neural Stem Cells
In this example, iPSCs were expanded in the NME7AB media which generates naïve stem cells that can be enzymatically passaged as single cells, expand 10X in four days, and are karyotypically stable. The cells then underwent neural induction using the PSC Neural Induction Medium from Thermo Fisher Scientific. Directed differentiation of naïve cells to neural stem cells increase efficiency by 150X over standard primed cells.
NME7AB increases final NSC yield compared to FGF based media. Data shows fold expansion during induction protocol was independent of starting cell number.
Cardiomyocytes
Sarcomeres are the basic unit of muscle to which myosin and actin attach and cause the contraction in cardiomyocytes. Immunofluorescent imaging of α-actinin shows that cardiomyocytes derived from naïve stem cells have a more organized sarcomere structure compared to those derived from FGF primed cells, which gives rise to more effective contractions.
Keys to success with naïve stem cells
- When first transitioning primed cells to naïve cells, it is ideal to have existing naïve stem cells to use as a reference/control. Minerva offers both male and female naïve stem cells lines
- Naïve stem cells should ideally be cultured under hypoxic conditions. (37°C, 5% CO2, 5% O2).
- Naïve stem cells have a much higher cloning efficiency than primed state cells, so it is important to monitor the cells closely and not to overgrow them. Passage cells at 75-85% confluence (~3-4 days).
- The cells grow in a monolayer and can be dissociated into single cells using TrypLE™
- NME7AB is replacement for FGF and is not compatible with FGF media, so NME7AB should not be added to other commercial products.
Conclusions
One of the most exciting aspects of induced pluripotent stem cells technology is the potential for generating differentiated cells for therapeutic uses such as Parkinson's disease and cardiac tissue damage. From past studies, we’ve identified some of the recurring challenges faced by translational researchers: spontaneous differentiation and insufficient number of the target differentiated cell type due to low yield, poor cell quality and functionality. NME7AB cultured naïve iPSCs do not spontaneously differentiate, have a much higher yield of directed differentiation than stem cells cultured in FGF-based media, and are of high quality and functionality. In addition, the cells are free of residual pluripotent cells as a result of our unique peptide that breaks the pluripotency signal and induces differentiation. The result is large quantities of high quality, biologically relevant cells.
References
1. A Primitive Growth Factor, NME7AB , Is Sufficient to Induce Stable Naïve State Human Pluripotency; Reprogramming in This Novel Growth Factor Confers Superior Differentiation.
2. Carter MG, Smagghe BJ, Stewart AK, Rapley JA, Lynch E, Bernier KJ, Keating KW, Hatziioannou VM, Hartman EJ, Bamdad CC, Stem Cells. 2016 Apr;34(4):847-59.
Mark G. Carter is group leader, stem cell biology, and Andrew K. Stewart is senior project manager at Minerva Biotechnologies.
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