Differentiation protocol for cranial neural crest-derived pericyte-like cells from human pluripotent stem cells

The successful differentiation of brain pericyte-like cells from human pluripotent stem cells may allow us to study their biological characteristics and their applications in the treatment of pericyte dysfunction-related neurodegenerative diseases, including ischemic stroke, Parkinson’s disease (PD) and so on. The protocol we present in the study provides a cranial neural crest originated, fast and robust forebrain pericyte-like cells differentiation method using either human embryonic stem cells or human induced pluripotent stem cells as a starting material. In vitro functional tests show contractile properties measured by gel contraction assay and carbachol treatment assay, vasculogenic potential measured by in vitro cord formation assay and Matrigel matrix implants and endothelial barrier function measured by transepithelial resistance (TEER).


Introduction
The blood-brain barrier (BBB) is a neurovascular unit (NVU) that serves as a physical and chemical barrier against plasma components, blood cells, and pathogens for protecting the central nervous system (CNS). The BBB also controls the exchange and movement of nutrients, hormones and other molecules into and out of the brain for proper functioning of the CNS 1 . Previous studies have demonstrated that the BBB consists of brain microvascular endothelial cells (BMECs), astrocytes, neurons, pericytes, and extracellular matrix around the vessels composed mainly of type IV collagen, bronectin, laminin, heparan sulfate, and perlecan 2, 3, 4 .
Pericytes play an essential role in the maintenance of the BBB and the dysfunction or degeneration of the pericytes contributes to the pathogenesis of diverse CNS disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, ischemic stroke and so on 1 . Therefore, pericyte replacement therapies may help to restore normal pericyte function and BBB integrity in these disorders. However, the scarcity of material sources greatly limits the application of primary pericytes in disease modeling and cell transplantation studies. Accordingly, human pluripotent stem cells (hPSCs) with the properties of self-renewal and pluripotency present an ideal cell model for the isolation of pericytes to study their development or the therapeutic effect in pericyte-related diseases. Although there are several protocols describing the derivation of pericytes from hPSCs, they are either time-consuming or may generate heterogeneous pericyte population with different origins 5,6 . Here, we successfully derive pericyte-like cells with cranial neural crest (CNC) origin from hPSCs (designated as hPSC-CNC PCs), which may represent an ideal cell source for the treatment of pericyte dysfunction-related disorders and help to model the human BBB in vitro for the study of the pathogenesis of such neurological diseases. N2B27-CDM contains DMEM/F12 supplemented with 1% N2, 2% B27, 1% L-Glu, 1% MEM minimum nonessential amino acids solution, 0.1% BSA, 0.1mM 2-Mercaptoethanol, 10 ng/ml bFGF, 50 U ml -1 penicillin and 50 mg ml -1 streptomycin. Store the medium at 4 °C and use it within 3 weeks.
-Pericyte induction medium: Pericyte induction medium contains PM supplemented with 50ng/mL PDGF-BB, 10ng/mL bFGF, 50 U ml -1 penicillin and 50 mg ml -1 streptomycin. Store the medium at 4 °C and use it within 3 weeks. 2. Remove the hPSC medium from the wells and wash with 1 X PBS twice.
3. Add a minimal amount of Accutase solution needed to cover the surface of the wells and incubate the plate at 37 °C 5% CO 2 for 5-10 min.
4. Gently triturate the cells to achieve a single cell suspension.
5. Dilute Accutase with 4 volumes of N2B27-CDM and centrifuge for 4 min at 1100rpm at room temperature.
6. Discard the supernatant and re-suspend the cells in N2B27-CDM containing 10μM ROCK inhibitor.
7. Plate the cells on Matrigel-coated 10-cm dish at a density of 10,000 cells per cm 2 and place the dish at 37°C 5% CO2 for 24 h. 8. On the next day, replace the medium with fresh NCN2 and culture for 6 days, during which the medium is changed every day.
hPSC-CNC PCs differentiation phase II: cranial neural crest cells isolation 9. On the day 6 or 7 in NCN2 culture, aspirate the medium from the dish and wash with 1 X PBS twice.
10. Add a minimal amount of Accutase solution needed to cover the surface of the dish and incubate the plate at 37 °C 5% CO 2 for 5 min.
11. Dilute Accutase with 4 volumes of 1 X PBS and gently triturate the cells. When cells are ready for plating, aspirate the solution and wash the ask three times with 1 X PBS. 20. Resuspend cranial neural crest cells with NCCM medium supplemented with 10 μM Y27632 and plate on culture ask pre-coated with PO/FN at a density of 10 5 cells/cm 2 . Incubate the ask at 37°C 5% CO 2 for 24h. 21. On the next day, replace the medium with pericyte induction medium and change the medium every 2-3 days.
22. When the cells are 80-90% con uent, remove the pericyte induction medium from the ask and wash with 1 X PBS twice.
23. Add a minimal amount of Accutase solution needed to cover the surface of the wells and incubate the plate at 37 °C 5% CO 2 for 1-2 min. 28. During pericyte induction, cells may be validated for pericyte markers using ow cytometric sorting.

Troubleshooting
Step 18: If small percentages of p75 bright HNK1 + cells were attained during cell sorting, it may be due to incorrect cell count in step 7. The cell density at the beginning of induction has a great in uence on the induction e ciency.
Step 21: if the cells do not attach to the culture plates or dishes, it may be due to poorly PO/FN coated or inadequate wash the PO/FN-coated cell culture ask with PBS. It is best to coat the ask overnight. Poly-L-ornithine is toxic to cells and make sure to wash the PO/FN-coated cell culture ask with 1 X PBS thoroughly.

Anticipated Results
The described protocol is able to generate a highly pure population of hPSC-CNC-derived pericyte-like cells.
Immuno uorescence staining shows extensive presence of pericyte-related markers such as NG2 and PDGFRβ.
In vitro functional tests show contractile properties measured by gel contraction assay and carbachol treatment assay, vasculogenic potential measured by in vitro cord formation assay and Matrigel matrix implants and endothelial barrier function measured by transepithelial resistance (TEER).