Clinically compatible differentiation protocol for human pluripotent stem cell-derived dopaminergic progenitor cells

doi:10.21203/rs.3.pex-954/v1

Method Article

Clinically compatible differentiation protocol for human pluripotent stem cell-derived dopaminergic progenitor cells

https://doi.org/10.21203/rs.3.pex-954/v1

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Cell replacement therapy with human pluripotent stem cells has the potential to be a new therapy for Parkinson’s disease (PD). This protocol induces human induced pluripotent stem cells (iPSCs) to dopaminergic progenitor cells (DAPs) as clinically compatible donor cells in 30 days. The protocol includes starting with high density culture, cell sorting by using a cell surface marker for floor plate, and a maturation culture to form floating aggregates. The DAPs differentiated with this protocol were used in a pre-clinical tumorigenicity and efficacy study aiming for approval to start a clinical trial in Japan.

Cell Biology

Stem cells

Neuroscience

Human pluripotent stem cells

Parkinson's disease

Cell transplantation therapy

dopaminergic progenitors

Parkinson’s disease (PD) is a neurodegenerative disease caused by a loss of dopaminergic (DA) neurons in the substantia nigra-striatal pathway. The neuronal cell death results in mainly motor symptoms including tremors, rigid muscles, slower movement, and impaired posture and balance. Cell therapy for PD is a concept in which the lost dopaminergic neurons are replaced by surgery. As donor cells, authentic neural progenitors with ventral mesencephalic (VM) phenotype are needed. Developmentally, dopaminergic neurons in substantia nigra are derived from fetal VM. Accordingly, aborted fetal midbrain tissues were used in clinical trials in the 1990s and showed some clinical benefit. Because fetal tissue is in short supply and its use has ethical issues, human pluripotent stem cells (PSCs) are being investigated as an alternative cell source for transplantation. There have been many reported protocols for mesencephalic DA induction from human PSCs at the laboratory level^1–4. Most of them cannot be applied to the clinic as they are, however. To meet the criteria of clinical application, xeno-materials and genetic modifications to the cells must be avoided, the prevention of contamination and GMP compatibility must be confirmed, and regulations with respective countries must be met. We have established a differentiation protocol for dopaminergic progenitor cells (DAPs) from human PSCs^{5, 6} and modified it for the purpose of a preclinical study and clinical application. The reagents and equipment are changed to clinical grade products to comply with the requirements by a Japanese regulatory agency.

Reagents

- Feeder-free human induced pluripotent stem cells (hiPSCs) cultured on a 90-mm dish

- iMatrix-511MG (Nippi, cat. no. 892005)

- D-PBS without Ca and Mg (nacalai tesque, 14249-24)

- TrypLE™ Select CTS™ (Thermo Fisher, cat. no. A12859)

- 0.5 mol/L-EDTA (pH8.0, nacalai tesque, 06894-14)

- CultureSure® DMSO (FUJIFILM Wako, cat. no. 031-24051)

- OTSUKA DISTILLED WATER (Otsuka Pharmaceutical, 1751)

- OTSUKA NORMAL SALINE (Otsuka Pharmaceutical, 0613)

- Glasgow's MEM (GMEM, Thermo Fisher, cat. no. 11710)

- KNOCKOUT^TM Serum Replacement for ESCs/iPSCs (KSR, Thermo Fisher, cat. no. 10828), 30 kGy gamma-ray irradiated

- MEM Non-Essential Amino Acids Solution (NEAA, Thermo Fisher, cat. no. 11140)

- Sodium Pyruvate, 100 mM (Sigma, S8636)

- StemSure® 10 mmol/L 2-mercaptoethanol solution (2-ME, FUJIFILM Wako, cat. no. 198-15781)

- CultureSure® 10 mmol/L Y-27632 solution, animal-derived free (FUJIFILM Wako, cat. no. 039-24591)

- Stemolecule™ LDN-193189 (REPROCELL, cat. no. 04-0074)

- CultureSure® A-83-01 (FUJIFILM Wako, cat. no.039-24111)

- FGF8, human, recombinant, animal-derived-free (FUJIFILM Wako, cat. no. 067-06231)

- Purmorphamine (FUJIFILM Wako, cat. no. 166-23991)

- CultureSure® CHIR99021 (FUJIFILM Wako, cat. no. 034-23103)

- Neurobasal® medium (1×), liquid -L-Glutamine (Thermo Fisher, cat. no. 21103)

- B27 supplement (50×) without vitamin A (Thermo Fisher, cat. no. 12587), 30 kGy gamma-ray irradiated

- Cell Therapy Systems GlutaMAX™- I CTS™ (100×), 200 mM (Thermo Fisher, cat. no. A12860)

- BDNF, human, recombinant, animal-derived-free (FUJIFILM Wako, cat. no. 028-16451)

- GDNF, human, recombinant, animal-derived-free (FUJIFILM Wako, cat. no. 070-06261)

- Ascorbic acid (TOWA Pharmaceutical)

- ACTOSHIN for injection (dibutyryl cyclic AMP, Daiichi-Sankyo Pharmaceutical)

- Gentacin® 40 (gentamycin, Takata Pharmaceutical)

- Fetal bovine serum gamma irradiated by SER-TAIN™ process, sourced in Australia (FBS, SAFC, cat. no. 12007C)

- D-glucose solution, 45% in H₂O, sterile-filtered (Sigma, cat. no. G8769)

- PE-anti-CORIN antibody (not commercially available) or human CORIN antibody (R&D, cat. no. MAB2209) and a fluorescent secondary antibody for cell sorting

Reagent setup

- 0.5×TrypLE Select solution

Ø Mix 90 mL of TrypLE Select CTS with 90 mL of D-PBS(-) and 90 μL of 0.5 mol/L-EDTA

Ø Store at room temperature and use within 6 months

- 8GMK media (8% KSR-GMEM)

Ø Mix 500 mL of GMEM with 45 mL of KSR, 5.6 mL of NEAA, 5.6 mL Sodium Pyruvate, and 5.6 mL of 2-ME

Ø Store at 5 °C and use within 1 month

- NB/B27 media

Ø Mix 500 mL of Neurobasal medium with 10 mL of B27 supplement without vitamin A and 5.1 mL of GlutaMAX™- I CTS™

Ø Store at 5 °C and use within 1 month

- LDN193189 reconstitution

Ø Add 4.515 mL of DMSO to 2 mg of Stemolecule™ LDN-193189 to obtain a concentration of 1 mM

Ø Mix well to completely reconstitute the powder

Ø Freeze the aliquots at -30 °C (stable for 6 months after reconstitution)