Novel stem cell growth factor NME7AB dramatically …...Novel stem cell growth factor NME7 AB...

Novel stem cell growth factor NME7 AB dramatically improves iPSC generation, expansion and differentiation Bamdad CC, Carter MG, Smagghe BJ, Stewart AK, Morse BP, Deary LT, Nash JSS, Page DM Despite the 10 years that have passed since the Nobel Prize winning discovery of iPSCs, their generation and expansion are still routinely done in bFGF-based media on feeder cells or extracellular matrix substrates. These methods are inefficient, labor-intensive and relatively non-scalable. We discovered a growth factor NME7 AB that is expressed in the inner cell mass of human blastocysts. iPSC generation using the Yamanaka factors in serum-free, FGF-free, NME7 AB media over an antibody adhesion layer increased feeder-free reprogramming efficiency by 1-2 orders of magnitude. The NME7 AB system enables single cell passaging and large-scale expansion in high-capacity flasks: 10-fold expansion in four days. Resultant cells express pluripotency markers, as well as naïve markers, have two active X chromosomes if generated from female cells and differentiate down all three germlines. Directed differentiation is superior to that of FGF grown cells in terms of quality, quantity and purity with no clonal differentiation bias. RESULTS METHODS Reprogramming We generated hiPSCs from human foreskin fibroblasts in a serum-free, feeder-free, FGF2-free media containing NME7 AB . We transduced fibroblasts with OCT4, SOX2, KLF4, and c-MYC (OSKM) in NME7 AB minimal media using Sendai virus. (100K human fibroblasts per well). Images 21-days post transduction Expansion We expanded the hiPSCs in MN-C3 coated T75 & T75 Flasks. Starting with two (2) 6-well plates, we expanded to 1.14 billion cells in 17 days Differentiation We formed EBs from the hiPSC lines and differentiated the cells 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 Feeder Based • High variability • Labor Intensive • 15% of colonies resulted in stable lines NME7 AB • Feeder-Free • High Efficiency • 86% of colonies resulted in stable lines 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 NME7 AB System provides high efficiency feeder-free reprogramming with improved cell line survival CONCLUSIONS This work demonstrates that NME7 AB is sufficient for long-term culture of human pluripotent stem cells in the absence of serum, feeder cells, FGF2 or other growth factors. iPSC generation in NME7 AB is more efficient, without the need for feeders, and have stable karyotypes. Human ES and iPS cells can be rapidly expanded in this culture system for high-efficiency directed differentiation. RESULTS Minerva Biotechnologies ♦ 40 Bear Hill Rd. Waltham, MA 02451 ♦ 781-487-0200 ♦ [email protected] Day 5 human blastocyst Day 3 human blastocyst Large-scale production of hiPSCs has been one of the major challenges for translational applications of hiPSC technology. Here we demonstrate a scalable, robust and economic method that yields over 1 billion high quality cells in 17 days. Cells expanded in the NME7 AB System maintain expression of pluripotency markers. These expanded NME7 AB -grown iPSCs were able to form embryoid bodies containing cells from all three germ layers. Day 4 10X Day 3 10X 20x 20x 20x 20x OCT4 NANOG SSEA - 4 TRA1 - 81 Pluripotency of the cells is also maintained in both 6-well and flask formats. OVERVIEW β-tubulin AFP Nestin 20x SMA 20x 20x 20x Day 5 human blastocyst NME7 Day 3 human blastocyst Ectoderm: Neuronal Rosette (x200) Mesoderm: Ossified Cartilage (x200) Endoderm: Gland (x200) NME7 AB -grown iPSCs form teratomas that generate all three germ lines.

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Transcript of Novel stem cell growth factor NME7AB dramatically …...Novel stem cell growth factor NME7 AB...

Page 1: Novel stem cell growth factor NME7AB dramatically …...Novel stem cell growth factor NME7 AB dramatically improves iPSC generation, expansion and differentiation Bamdad CC, Carter

Novel stem cell growth factor NME7AB dramatically improves iPSC generation, expansion and differentiationBamdad CC, Carter MG, Smagghe BJ, Stewart AK, Morse BP, Deary LT, Nash JSS, Page DM

Despite the 10 years that have passed since the Nobel Prize winning discovery of iPSCs, their generation and expansion are still routinely done in bFGF-based media on feeder cells or extracellular matrix substrates. These methods are inefficient, labor-intensive and relatively non-scalable.

We discovered a growth factor NME7AB that is expressed in the inner cell mass of human blastocysts. iPSC generation using the Yamanaka factors in serum-free, FGF-free, NME7AB media over an antibody adhesion layer increased feeder-free reprogramming efficiency by 1-2 orders of magnitude.

The NME7AB system enables single cell passaging and large-scale expansion in high-capacity flasks: 10-fold expansion in four days. Resultant cells express pluripotency markers, as well as naïve markers, have two active X chromosomes if generated from female cells and differentiate down all three germlines. Directed differentiation is superior to that of FGF grown cells in terms of quality, quantity and purity with no clonal differentiation bias.

RESULTS

METHODSReprogrammingWe generated hiPSCs from human foreskin fibroblasts in a serum-free, feeder-free, FGF2-free media containing NME7AB. We transduced fibroblasts with OCT4, SOX2, KLF4, and c-MYC (OSKM) in NME7ABminimal media using Sendai virus. (100K human fibroblasts per well). Images 21-days post transductionExpansionWe expanded the hiPSCs in MN-C3 coated T75 & T75 Flasks. Starting with two (2) 6-well plates, we expanded to 1.14 billion cells in 17 daysDifferentiationWe formed EBs from the hiPSC lines and differentiated the cells into all three germ lineagesKaryotype StabilityThe cells were confirmed to have a normal karyotype by cytogenetic analysis performed on twenty G-banded metaphase cells

Feeder Based• High variability• Labor Intensive• 15% of colonies resulted in stable lines

NME7AB• Feeder-Free• High Efficiency• 86% of colonies resulted in stable lines

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 NME7AB System provides high efficiency feeder-free reprogramming with improved cell line survival

CONCLUSIONSThis work demonstrates that NME7AB is sufficient for long-term culture of human pluripotent stem cells in the absence of serum, feeder cells, FGF2 or other growth factors. iPSC generation in NME7AB is more efficient, without the need for feeders, and have stable karyotypes. Human ES and iPS cells can be rapidly expanded in this culture system for high-efficiency directed differentiation.

RESULTS

Minerva Biotechnologies ♦ 40 Bear Hill Rd. Waltham, MA 02451 ♦ 781-487-0200 ♦ [email protected]

Day 5 human blastocystDay 3 human blastocyst

Large-scale production of hiPSCs has been one of the major challenges for translational applications of hiPSC technology. Here we demonstrate a scalable, robust and economic method that yields over 1 billion high quality cells in 17 days.

Cells expanded in the NME7AB System maintain expression of pluripotency markers.

These expanded NME7AB-grown iPSCs were able to form embryoid bodies containing cells from all three germ layers.

Day 410X

Day 310X 20x 20x

20x20x

OCT4 NANOG

SSEA-4 TRA1-81

Pluripotency of the cells is also maintained in both 6-well and flask formats.

OVERVIEW

β-tubulin

AFPNestin

20x