Allen Chen, Yu Ming Lim, Shaul Reuveny and Steve Oh

Transcription

1 EXPANSION AND DIFFERENTIATION OF HUMAN PLURIPOTENT STEM CELLS TO NEURAL PROGENITORS: A SIMPLIFIED BIOREACTOR PROCESS REPLACING NOGGIN WITH 2 SMALL MOLECULES Allen Chen, Yu Ming Lim, Shaul Reuveny and Steve Oh

2 BTI Stem Cell Bioprocessing Platform Expansion Process development and optimization Expansion Mesenchymal Stem Cells Microcarrier Differentiation Osteoblast Chondrocyte Differentiation Neural progenitor Cardiomyocyte Hemangioblast Material science ECM coating Microcarrier synthesis and characterization Pg 2

3 Outline 1. Expansion of human pluripotent stem cells on microcarriers 2. Differentiation of human pluripotent stem cells to neural progenitors PSA-NCAM + (polysialylated neuronal cell adhesion molecule ) neural progenitors Comparison of two protocols Differentiation to dopaminergic neurons Pg 3

4 Expansion and differentiation of human pluripotent stem cells (hpscs) to neural progenitors Reasons for using microcarrier technology Microcarriers Tissue culture flask Scalability Yes Yes, but limited Production capacity Higher cell yield per volume Low cell yield per volume Process control Comprehensive Limited Material requirement Lower High Handling Simplified More labor intensive SEM micrograph showing hpscs on microcarriers Bioreactors from Sartorius NUNC tissue culture flask Pg 4

5 2D Cell Density (x10 6 cells/ml) Microcarrier - Static Microcarrier - Spinner Human Pluripotent Stem Cell (hpscs) Expansion on Microcarriers in Stirred Bioreactor 4 3 Microcarrier - Spinner Microcarrier - Static 2D 2 * * * * * * Time (Day) Stirred Microcarrier culture Static Microcarrier culture 2D culture Bardy J, A Chen, YM Lim, S Wu, S Wei, H Weiping, K Chan, S Reuveny and SKW Oh. (2013). Microcarrier suspension cultures produce high yields of neural progenitor cells from human pluripotent stem cells. Tissue Engineering Part C: Methods 19:

6 Human Pluripotent Stem Cell Expansion on Microcarriers in Stirred Bioreactor Higher cell yield and pluripotency achieved in double feeding regime Bardy J, A Chen, YM Lim, S Wu, S Wei, H Weiping, K Chan, S Reuveny and SKW Oh. (2013). Microcarrier suspension cultures produce high yields of neural progenitor cells from human pluripotent stem cells. Tissue Engineering Part C: Methods 19: Pg 6

7 Differentiation of human pluripotent stem cells to neural progenitors Microcarrier Platform hpsc Expansion Induction of Neural differentiation via Advantages of Small molecules versus Recombinant Protein More economical Minimal lot-to-lot variabilities Longer shelf lives Recombinant protein*: Noggin 500ng/ml 2 small molecules # : - Dorsomorphin 5uM - SB uM Day 0 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Noggin Protocol Small molecules Protocol KO media N2B27 + Noggin N2B27 + bfgf + EGF KO media + SM NIM media + SM N2B27 + bfgf + EGF * Bardy J, A Chen, YM Lim, S Wu, S Wei, H Weiping, K Chan, S Reuveny and SKW Oh. (2013). Microcarrier suspension cultures produce high yields of neural progenitor cells from human pluripotent stem cells. Tissue Engineering Part C: Methods 19: # Kim D-S, J Lee, J Leem, Y Huh, J Kim, H-S Kim, I-H Park, G Daley, D-Y Hwang and D-W Kim. (2010). Robust Enhancement of Neural Differentiation from Human ES and ips Cells Regardless of their Innate Difference in Differentiation Propensity. Stem Cell Reviews and Reports 6: Pg 7

8 Cell density (x106 cells/ml) Differentiation of human pluripotent stem cells to neural progenitors % PSA-NCAM positive cells Day 0 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13 Noggin Protocol Small molecules Protocol KO media N2B27 + Noggin N2B27 + bfgf + EGF KO media + SM NIM media + SM N2B27 + bfgf + EGF 18 Small molecules Recombinant protein Time (day) Time (day) Small molecules generate higher cell growth Earlier induction of PSA-NCAM positive NPCs Protocol Overall Cell Yield Noggin NPC/ml 163 NPC/hPSC seeded Small Molecules NPC/ml 398 NPC/hPSC seeded

9 Characterization of hpsc derived Neural Progenitors H&E staining of neural progenitors on microcarriers Red arrow indicates microcarrier

10 Characterization of hpsc derived Neural Progenitors Positive expression of typical neural progenitor markers DAPI (Nuclei) PAX6 Nestin White arrows indicated microcarriers Multipotency of neural progenitors 100 µ m ßIII-Tubulin (Neuron) GFAP (Astrocyte) O4 (Oligodendrocyte) Pg 10

11 Differentiation of hpsc derived neural progenitors to dopaminergic neuron differentiation on microcarriers Spontaneous differentiation Directed differentiation Spontaneous differentiation p-value < 0.01 Directed differentiation p-value < Purity of Dopaminergic Neuron (%) Dopaminergic Neuron Yield (million cells/ml) MAP2 + MAP2 + Neurons MAP2 + TH + DA neurons TH + Tyrosine hydroxylase βiii-tubulin DAPI White Arrows indicate microcarriers

12 Conclusion Microcarrier based cultivation for human pluripotent stem cells is superior than 2D culture platform. A typical cell yield of highest cell yield cells/ml and 15 fold expansion is obtained. A higher cell yield of cells/ml can be obtained by increase the medium feed frequency. Improved neural differentiation efficiency by replacing Noggin with small molecules (Dorsomorphin and SB ): Small molecules are more cost effective Earlier onset of NPC expression Higher cell growth NPCs/ml (398 NPCs/hPSC seeded) as compared to NPCs/ml (163 NPCs/hPSC seeded) Maintain multi-lineage differentiation potential Differentiation to dopaminergic neuron on microcarriers Pg 12

13 Acknowledgement Funding from the Agency for Science Technology and Research, A*STAR And the Joint Council Office, A*STAR Pg 13