Mapping the Biochemical Landscape of Human Cortical Induced Pluripotent Stem Cells Brain Organoid Maturation Using Label-Free Raman Imaging

In fifteen words or less, explain the significance of this contribution (Novel Aspect).: First study chemically imaging human iPSC as a novel model for future neurodevelopmental diseases

Abstract Text:

The human brain is an extraordinarily complex organ, and understanding its neurodevelopmental processes and disease mechanisms has been hindered by limited access to living tissue. However, recent advancements in human induced pluripotent stem cell (hiPSC) technology have enabled the generation of 3D brain organoids, offering a promising in vitro model for brain research. In this study, we develop brain organoids and employ label-free Raman microscopy to image and evaluate its biochemical composition at various stages of organoid maturation.

Two sets of human healthy brain organoids were derived from iPSCs (CS5nXHi-CTR) and cultured over a period ranging from day 3 to 6 months. Organoid slices, sectioned to 10 µm thickness, were analyzed at 10 distinct timepoints. A custom-built Spontaneous Raman (SR) system was used to collect high-resolution images over a spectral range of 200 cm⁻¹ to 3200 cm⁻¹, with a 1.3 µm step size and a 150x180 pixel resolution covering a scan area of 196x234 µm. A 660 nm laser with a 50x objective lens, 35 mW laser power, and a 0.3 s exposure time was used to collect over 1.2 million spectra from 42 images. Key maturation stages (2, 3, 4 and 6 months) were also imaged with a finer 5 µm step size, to capture gradients in biochemical components of the cortical landscapes, with a field of view (FOV) of 750x900 µm. Data preprocessing included background subtraction using the EMSC algorithm, baseline correction, and normalization to harmonize the dataset.

Preliminary Multivariate Curve Resolution (MCR) analysis enabled the identification of key biomolecular components, including lipids (2853, 1438, and 1662 cm⁻¹), proteins (2935, 1662, 1450, 1004, and 850 cm⁻¹), and nucleic acids (2971 and 785 cm⁻¹). These components facilitated label-free imaging of organoid development, revealing critical maturation features: ‘rosette-like’ structures emerged at day 15, marking the onset of neurogenesis. At later timepoints (e.g., 6 months) lipid gradients appeared within cortical regions, consistent with the expected maturation of glial populations. These findings highlight the potential of label-free Raman imaging for mapping the biochemical maturation of brain organoids, opening new avenues for studying neurodevelopmental diseases.