Significant Improvement in Feeder-free Generation, Expansion and Differentiation of iPSCs Using Novel Stem Cell Growth Factor

By on April 3, 2017

In this poster, researchers at Minerva Biotechnologies describe their discovery of a novel growth factor, NME7AB, and its use in the AlphaSTEM® Culture System. NME7AB is expressed in the inner cell mass of the human blastocyst. By using this growth factor with Yamanaka factors in AlphaSTEM™ Naïve hPSC Medium over an antibody culture substrate, the authors present data demonstrating an increase in iPSC feeder free reprogramming efficiency by 1-2 orders of magnitude.

The authors show that using the NME7AB based AlphaSTEM® Culture System , enabled single cell passaging and large scale expansion in high capacity flasks. They were able to achieve 10-fold expansion in just 4 days. Stem cell expansion is faster and scalable, and can be completely automated since it eliminates unwanted spontaneous differentiation.

Furthermore, cells cultured in these conditions expressed pluripotency markers as well as naïve markers, had two active X chromosomes (if generated from female cells) and differentiated down all three germ lines. They also found that using this method, directed differentiation was superior to that of FGF grown cells in terms of quality, quantity, and purity with no clonal differentiation bias.

Methods

Reprogramming

Authors generated hiPSCs from human foreskin fibroblasts in a serum-free, feeder-free, FGF2-free media containing NME7AB. Then transduced fibroblasts with OCT4, SOX2, KLF4, and c-MYC (OSKM) in NME7AB minimal media using Sendai virus with 100,000 human fibroblasts per well.

Expansion

hiPSCs were expanded in MN-C3 coated T75 & T75 Flasks. Starting with two 6-well plates, cells were expanded to 1.14 billion cells in 17 days.

Differentiation

Embryoid bodies were formed from the hiPSC lines and cells were differentiated into all three germ lineages.

Karyotype Stability

The cells were confirmed to have a normal karyotype by cytogenetic analysis performed on twenty G-banded metaphase cells.

Results

Highlights from the results section of the poster include:

High efficiency feeder-free reprogramming

The AlphaSTEM® Culture System enabled high efficiency feeder-free
 reprogramming with improved cell line survival, thus reducing the variability and labor required with Mouse Embryonic Fibroblast cells (MEFs) (Figure 1).

Figure 1:

 

Cells Maintain Expression of Pluripotency Markers

Cells expanded in the AlphaSTEM® Culture System maintained expression of pluripotency markers (Figure 2).

Figure 2:

Embryoid bodies were formed and cells were differentiated into all three germ lineages

iPSCs expanded using AlphaSTEM® were able to form embryoid bodies containing cells from all three germ layers (Figure 3).

Figure 3:

 

Large-scale Production of hiPSCs

Authors address the challenge of large-scale production of hiPSCs by demonstrating an economically scalable process that yields over 1 billion high quality cells in 17 days (Figure 4).

Figure 4:

 

For more data and full details of the highlights discussed here, please click on the poster below to view in full size.

For more information on ordering AlphaSTEM® please visit: www.minervabio.com.

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