Induced-pluripotent stem cells (iPSC) are produced from a variety of source tissues including fibroblasts, epithelial progenitor cells, peripheral blood mononucleocytes (PBMC), and others. For Human iPSC, the pluripotency state is sometimes referred to as the “primed” state. Among iPSC lines, heterogeneity has been shown to exist in proliferation and the capacity to differentiate. This can cause issues in data interpretation or even limit the utility of Human iPSC in some disease cell models for basic research or drug discovery.
Recent publications have shown that Human iPSC, when cultured under special conditions, can be transitioned to what may be a more primitive form perhaps similar to cells in the pre-implantation state of a developing embryo. This state is referred to as the “naïve” state. In mouse and rabbit iPSC systems, naïve state cells have been shown to increase the efficiency and reproducibility (less bias) of terminal differentiation, including giving rise to some terminally differentiated cell types that display greater maturity. Thanks to this evidence (in other species), the accelerated growth characteristics of naïve cells, and other attributes linked to pluripotency, there is worldwide a rapidly growing interest in the Human stem cell research community.
ReproNaïve Cell Culture Medium
ReproCELL’s ReproNaïve™ Culture Medium is the first commercially available formulation designed to transition Human iPSC (primed state) to the “naïve” state. The medium uses a special combination of proprietary inhibitors, and hypoxic culture conditions (5% O2), to grow and passage iPSC on mouse SL10 feeder cells to achieve the naïve state.
ReproNaive™ Culture Medium will help to accelerate basic research on stem cells and disease model systems. Currently, most of the research with naïve cells is being done in non-profit basic research labs. However, as the base of knowledge grows concerning this biological phenotype, it is likely there will be a significant impact on Regenerative Medicine in the future.
Naïve state cells generally exhibit expression of the same pluripotency markers including Oct3/4, Nanog, Tra1-81, and SSEA4. However, KLF2 and KLF4 expression levels are markedly higher and TRE3 expression shows greater nuclear localization.
Furthermore, in female-derived iPS cell lines, expression of XIST (X-inactive specific transcript) is down-regulated indicating some reactivation of the dormant X-chromosome. When grown in ReproNaive™ medium, iPS cell colonies can be generated from single cells and they exhibit a compact dome-shaped morphology. Growth rates are typically much faster than the original primed state cells (e.g. 15 hr versus 25 hr doubling times), allowing for up to 100-times more cells to be generated in a typical day culture. Furthermore, ReproNaive™ -derived naïve state cells have been directed into neuronal cell differentiation with high efficiency, and also shown to spontaneously differentiate into all three germ-cell layers. An area of keen interest and investigation is whether naïve state hiPSC exhibit less differentiation bias or can give rise to more mature terminally differentiated cell types, as has been shown with rabbit and mouse naïve state iPS.
Culture of Human pluripotent stem cells in ReproNaïve medium enables stem cell researchers to generate and maintain naïve cells without the time-consuming need for genetic modification.
- Enables efficient conversion of Human primed, pluripotent stem cells to naïve stem cells that maintain naïve stem cell properties and pluripotency
- Maintains the flat colony morphology and rapid doubling time associated with naïve cells (Figures 1, 2,4)
- Promotes expression of markers with subcellular localization expected for naïve stem cells (Figure 3)
- Retains cell ability to differentiate into all three germ layers (Figure 5)
- Demonstrates naïve stem cell DNA hypermethylation consistent with the conversion from the prime to the naïve state
- Provides convenient, cost effective maintenance of naïve cells using a simple to prepare medium
- Requires fewer media supplements to buy and prepare
- Maintains naïve state for >15 passages
- Provides shorter time to achieve naïve stem cell culture
- Direct conversion without the need for genetic modification steps
Successful conversion to the naïve state requires the use of hypoxic culture (low oxygen) conditions. The medium needs to be reconstituted by the addition of a supplement supplied with the product, in addition to Human recombinant LIF which is not included in the kit. The supplement contains an inhibitors cocktail (proprietary) and is supplied in 5 aliquots (0.7 ml), each suitable for preparation of 100 ml of medium. Upon reconstitution, the media is stable for approximately 2 weeks. Addition of Y27632 (Rock Inhibitor) for the first 48 hours after passage is also recommended.
|ReproNaïve™ Cell Medium|
|ReproNaïve™ Cell Medium||500 mL Medium + supplement||RCHEMD008|
|Stemfactor™ Activin A, Human Recombinant||5 μg||03-0001|
|Stemfactor LIF, Human Recombinant||10 μg||03-0016|
|Stemolecule™ IM-12||2 mg||04-0081|
|Stemolecule PD0325901||2 mg||04-0006|
|2 mg, DMSO solution||04-0006-02|
|Stemolecule SB590885||2 mg||04-0080|
|Stemolecule WH-4-023||2 mg||04-0079|
|Stemolecule Y27632||2 mg||04-0012|
|2 mg, DMSO solution||04-0012-02|
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