Derivation of a Human Brain Organoid with Microglia Development

We present a protocol to generate a human brain organoid containing resident microglia and neurons by incorporating induced pluripotent stem cell-derived hematopoietic progenic cells into the neuronal organoid induction and development process. This method provides a model to study the interactions between microglia and neurons during human brain development. 3D brain organoids are mini brains generated from induced pluripotent stem cells or iPSCs.

As an alternative to animals and human subjects, they have been widely used in mimicking human brains in the research of brain development and neurologic disorders. They are great tools for advancing human health research. Conventional protocols for 3D human brain organoids rarely generate microglia, the resident immune cell of the human brain, as these cells arise from a different embryonic lineage than neurons.

While microglia can be added to brain organoids at a later stage of differentiation, a method to better mimic human brain development is needed. Most other protocols add HPCs or microglia at the end of cerebral organoid generation. We generate embryoid bodies from mixed iPSCs and HPCs before neural induction.

This may better mimic human brain development as microglia precursors migrate to the brain very early in development. Additionally, using HPCs saves total differentiation time as microglia differentiation protocols often take over 30 days. Therefore, our protocol can save time and reagent costs.

To begin, culture dissociated human-induced pluripotent stem cells or iPSCs in a 12-well plate containing one milliliter of E8 Flex medium, incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, check the colonies under the microscope and count the number of colonies by scanning all fields in a well. Replace the medium with one milliliter of Medium A from the hematopoietic kit.

Place the plate back into the incubator for 48 hours. On day three, replace 500 microliters of the spent medium with 500 microliters of fresh Medium A, and incubate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The next day, observe cell growth and differentiation from the iPSC colonies.

Replace the medium with one milliliter of Medium B.Monitor cell differentiation under the microscope and perform a half medium change with 500 microliters of Medium B every other day for six to seven days. On day 10, observe the presence of bright single round cells with the morphology of normal hematopoietic progenitor cells, or HPCs, floating or loosely attached to the flat layer cell with some loose aggregates. Next, collect all the differentiated HPCs by pipetting up and down three times with a one-milliliter pipette tip.

Transfer the cells into a 15-milliliter tube. Centrifuge the cell suspension at 300G for five minutes and resuspend the pellet in one milliliter of Medium B.Add 20 microliters of cell suspension into a cell-counting chamber and count the cells using a cell meter. Adjust the cell concentration to 1 million cells per milliliter using Medium B.The serial dilution culture technique produced appropriate numbers and sizes of iPSC colonies with colonies showing significant morphological changes by the end of culture in Medium A.After three days in Medium B, HPC differentiation was observed with the appearance of homogenous round cells.

At day 10, the HPCs predominantly formed colony-like clusters without significant debris. After collecting HPCs differentiated from human iPSCs, verify that an iPSC's culture has reached 80%confluency. Add 500 microliters of Anti-Adherence Rinsing Solution into one well of the micro well plate, minimizing bubble formation.

Spin the plate at 2, 000G for five minutes in a swinging bucket rotor fitted with plate holders. Remove the rinsing solution completely and wash the wells twice with one milliliter of DPBS and then with one milliliter of DMEM/F-12 without generating bubbles. Treat the iPSCs with one milliliter of ACCUTASE solution to initiate cell dissociation.

Observe the cells under the microscope, noting signs of cell separation while cells remain attached to the bottom. Replace the ACCUTASE solution with one milliliter of DMEM/F-12 medium, then, mix up and down to dissociate the cells into single cells. Transfer the dissociated cells into a 15-milliliter tube and add DMEM/F-12 medium to reach a final volume of five milliliters.

Centrifuge the cells at 300G for five minutes and resuspend the pellet in one milliliter of E8 Flex medium. Count the cells to adjust the cell concentration to 1 million cells per milliliter in the E8 Flex medium. Next, mix iPSCs with HPCs in a ratio of two to one into the well of the micro well culture plate.

Add one microliter of ROCK Inhibitor Y-27632 stock solution per one milliliter of medium into the supernatant and shake the plate side to side. Spin the plate at 300G for five minutes and observe under the microscope to confirm that cells have settled into the micro wells. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide.

After 48 hours, observe the cells under the microscope for embryoid bodies, or EBs, formation. Next, treat a 24-well cell culture plate with 500 microliters of Anti-Adherence Rinsing Buffer for 15 minutes to create a low attachment plate. Wash the wells twice with one milliliter of DPBS and add one milliliter of E8 Flex medium to each well.

Using a one-milliliter-wide-orifice pipette tip, resuspend the EBs in the micro well culture plate. Then, for each well of the low-attachment 24-well plate, add 100 microliters of medium containing the EBs. Incubate the plate in a 37-degree Celsius and 5%carbon dioxide incubator for 48 hours.

To begin, thaw the aliquots of the basement membrane matrix on ice for 30 minutes. On day two after EB formation, using a pre-chilled 20-microliter pipette tip, add 15 microliters of ice-cold basement membrane matrix in small drops on top of the medium to coat the EBs. Place the plate in a 37-degree Celsius and 5%carbon dioxide incubator for 48 hours.

On day four after EB formation, carefully remove 500 microliters of medium without disturbing the EBs and repeat basement membrane matrix coating as demonstrated previously. Then, add 500 microliters of pluripotent stem cells neural induction medium on top of the cells. Place the plate in a 37-degree Celsius and 5%carbon dioxide cell incubator.

On day six and eight, perform a half-medium change by carefully replacing 500 microliters of medium with 500 microliters of fresh neural induction medium. After incubation on days 10, 12, and 14, perform a half-medium change with neural stem cell medium. On day 15, transfer the well-containing spheres, along with the medium, to a new 12-well plate treated with Anti-Adherence Rinsing Solution.

Add 500 microliters of neuronal maturation medium on top of the culture. During the maturation stage, monitor the medium for signs of nutrient depletion such as a color change. If nutrient depletion is observed, transfer the organoid to a six-well plate.

On day 17, supplement the maturation medium with cytokines for six days to facilitate microglial maturation. Finally, on day 23, collect the resulting neural organoids for characterization. High-quality HPC and iPSC mixtures formed EBs within 24 hours showing continuous growth and minimal debris.

After transfer to a new plate, EBS continued to grow until reaching a plateau in the maturation medium.