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Stem Cell Technology

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Naive Stem Cell Culture

Minerva Biotechnologies is the first company to generate human naïve state induced pluripotent stem cells (iPS cells) using a single, naturally occurring human stem cell growth factor. Scientists believe that because these earlier stem cells have a clean slate, they are more easily directed to develop into functional mature cells, which could be used for transplant.

Naïve stem cells have several advantages over current stem cells, called ‘primed’ state cells, which are important for the future of stem cell therapies. Naïve stem cells do not yet have DNA methylation marks that commit the cells to certain developmental decisions. Additionally, naïve stem cells have a much higher cloning efficiency than primed state cells, which will be critical for the realization of stem cell based gene therapies.

A.         B. 

  • A. X- chromosome inactivation in Naïve cells - Cells grown in FGF (left) are showing X chromosome inactivation, where the cells grown in AlphaSTEM have no X inactivation, typical of naïve cells.
  • B. Naïve cells have a unique genetic profile - RNA sequencing shows a completely different genetic fingerprint between cells grown in bFGF and cells grown with AlphaSTEM.

A superior alternative to FGF-based cell culture

Currently, many stem cell culture scientists work with cells that require LIF and/or bFGF and numerous small molecules, such as demethylases and MAP kinase inhibitors, to keep their stem cells in a naïve-like state.  While this achieves similar cell culture conditions to naïve stem cells, there are significant differences in gene expression profiles of naïve-state markers, a decrease in differentiation potential, and unstable karyotypes.  

Using Minerva’s AlphaSTEM™ Culture System, iPSC generation is simpler and orders of magnitude more efficient, cell expansion is faster, scalable, and can be completely automated since it eliminates unwanted spontaneous differentiation while maintaining a normal karyotype, and differentiation efficiency is increased with improved yield, cell quality, and functionality. 

The AlphaSTEM™ Culture System includes the following products:

  • AlphaSTEM™ Naive hPSC Medium: Serum-free medium for the generation and expansion of naïve pluripotent stem cells.
  • AlphaSTEM™ Culture Substrate: Cell attachment antibody that supports generation and expansion of naive pluripotent stem cells.  
  • AlphaSTEM™ Differentiation Inducer: Synthetic peptide that competitively inhibits the interaction of NME7AB leading to increased efficiency of directed differentiation and ensures the elimination of residual pluripotent cells.

Enhanced Feeder-Free Reprogramming using AlphaSTEM™


Sendai Virus / 100K human fibroblasts per well / 21-days post transduction

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. The AlphaSTEM™ System provides high efficiency feeder-free reprogramming with improved cell line survival.

A more efficient expansion system

Doubling times of naïve stem cells are faster than primed cells cultured in bFGF.  Using AlphaSTEM, stem cell expansion is faster, scalable and can be completely automated since it eliminates unwanted spontaneous differentiation, while maintaining a normal karyotype.

  • One single naturally occurring growth factor
  • No spontaneous differentiation
  • Enzymatically pass as single cells
  • 10-20-fold expansion in 4 days
  • Karyotype stable > 70 passages
  • iPS generation 20X more efficient than in FGF media
  • No clonal restriction; every iPS clone able to form cardiomyocyte, hepatocytes, neural progenitors
  • Better differentiation than primed state stem cells


10X- 20X Expansion in 4days

Enhanced purity and cell function

Directed differentiation efficiency and functionality are increased with ‘clean slate’ AlphaSTEM naïve cells.  The use of the AlphaSTEM™ Differentiation Inducer further improves these benefits.  The AlphaSTEM™ Differentiation Inducer is a synthetic peptide that competitively inhibits the interaction of NME7AB and the MUC1* growth factor receptor that promotes pluripotency leading to increased efficiency of directed differentiation and ensures the elimination of residual pluripotent cells. AlphaSTEM™ Differentiation Inducer is added only once when differentiation is desired.The addition of AlphaSTEM™ Differentiation Inducer increases yield of directed differentiation, synchronizes differentiation and ensures the elimination of residual pluripotent cells.

Example: Improved Cardiomyocyte Contraction

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 AlphaSTEM cultured naïve stem cells have a more organized sarcomere structure compared to those derived from FGF2 primed cells, which gives rise to more effective contractions. 

Platform Benefits

  • A single naturally occurring growth factor
  • Feeder-free iPSC generation that is simpler and orders of magnitude more efficient the FGF based methods
  • Stem cell expansion that is faster, scalable and can be completely automated since it eliminates unwanted spontaneous differentiation, while maintaining a normal karyotype
  • 10-20-fold expansion in 4 days
  • Karyotype stable > 70 passages
  • No clonal restriction; every iPS clone able to form;cardiomyocyte, hepatocytes, neural cells
  • Superior differentiation with improved yield, cell quality, and functionality
     

Purchase Reagents

 

Publication:
A Primitive Growth Factor, NME7AB, Is Sufficient to Induce Stable Naïve State Human Pluripotency; Reprogramming in This Novel Growth Factor Confers Superior Differentiation