Neural iPSC unit (NIPS) has been recently established in co-operation with Jari Koistinaho's lab at the Neuroscience Center (Biomedicum 1, 2nd floor, Meilahti Campus) as a part of the Biomedicum Stem Cell Center (BSCC) core facility and Understanding Human Brain (UHBRAIN) profiling area of the University of Helsinki. We provide brain cell differentiation services using state-of-the art methods in our well-equipped cell culture laboratory. Co-cultures of our iPSC-derived neurons and astrocytes produce functional neuronal networks that develop synchronized electric activity. We also provide consultation and training services. The descriptions of cell differentiation protocols we use are listed below.
The differentiation is based on the protocol by Shi et al., 2012, and generates over 90% glutamatergic VGLUT1-positive cortical neurons. It takes 3-4 weeks to get neural progenitor cells from iPS cells, approx. 4-8 million cells per differentiation. It takes another 5 weeks to obtain functionally mature neurons. Co-culture with astrocytes is usually necessary to obtain functional maturation (network activity).
The differentiation is based on the protocol by Nehme et al., 2018 and combines the use of tetracycline-inducible NGN2-encoding lentiviral vector and dual SMAD/WNT inhibition. The protocol yields glutamatergic neurons with features of cortical layer II-III neurons in just 7 days (2-4 million cells per differentiation). It takes 4-5 weeks in co-culture with astrocytes for the neurons to achieve functional maturity (synchronous network activity).
Differentiation is based on the protocols by Nat et al., 2007, and Hicks et al., 2009, and generates over 90% glutamatergic VGLUT1-positive cortical neurons. Neuronal progenitors are expanded as spheres in the presence of FGF2 growth factor for 1,5 – 2 months. Thereafter the spheres are dissociated, and the neurons are maturated on poly-D-lysine/laminin-coated plates for another 1-2 weeks in the presence of growth factors BDNF and GDNF. One set of differentiation can generate 4-10 million cells. Co-culture with astrocytes for 4-8 weeks is usually necessary to achieve functional maturation (network activity). This protocol has been extensively used by Jari Koistinaho’s lab (Tiihonen et al., 2020; Tiihonen et al., 2019; Oksanen et al., 2017).
The differentiation is based on the protocol by Krencik et al., 2011, and generates GFAP/S100β-positive astrocytes. Glial progenitors are expanded as spheres in the presence of growth factors FGF2 and EGF for 5 – 7 months. Thereafter the spheres are dissociated, and the astrocytes are maturated on Matrigel-coated plates for another week in the presence of growth factors BMP4 and CNTF. One set of differentiation can generate 4-10 million cells. This protocol has been used extensively used by Jari Koistinaho’s lab (Koskuvi et al., 2022; Sonninen et al., 2020; Tiihonen et al., 2020; Oksanen et al., 2017).
The differentiation is based on the protocols by Abud et al., 2017, and McQuade et al., 2018, and generates Iba1/P2Y12/CX3CR1-positive microglia. Hematopoietic progenitors are first generated using commercial STEMdiff Hematopoietic kit (StemCell Technologies). Thereafter, hematopoietic progenitors are differentiated into microglia using the combination of growth factors IL-34, MCSF and TGFβ. Microglia are then further maturated for 4 days using in the presence of neuronal factors CD200 and CX3CL1. One 6-well plate containing 420 000 progenitors will generate 2.4-3 million of mature microglia.
The differentiation is based on the protocol by Lancaster et al., 2014. IPSCs are dissociated into a single-cell suspension and plated on a 96-well U-shaped low-attachment plate. When nice round embryoid bodies with translucent outer layer (neuroectoderm) are formed, they are embedded into Matrigel and transferred to an ultra-low attachment 6-well plate on an orbital shaker for further differentiation. At 2 months, cerebral organoids typically exhibit multiple ventricular zones with proliferating progenitors (PAX6+, SOX2+). Ventricular zones are surrounded by cortical plate-like areas containing maturating neurons (MAP2+, TBR1+). CTIP2+ deep layer neurons appear earlier, and SATB2+ superficial layer neurons later in the differentiation process. GFAP+ astrocytes appear only after 3 months of the differentiation process. One 96-well plate would produce approx. 50 good-quality organoids.
The service includes plating and immunostaining iPSCs for 4 different pluripotency markers: OCT4, Nanog, SSEA4, and Tra1-81. The service also includes RNA isolation and RT-qPCR reactions for 4 different pluripotency genes: OCT3/4, NANOG, SOX2, and LIN28 plus GAPDH as a control gene.
We have Axion Maestro Edge multiwell microelectrode array system with full temperature and CO2 control. The system allows for the use of Axion 24-well and 6-well plates. Simultaneous measurement of the signal in all the wells and electric stimulation are possible. The system is equipped with software for the quantification of spikes, bursts, and network bursts. Raw data can also be exported and analyzed elsewhere.