A protocol for the generation of EPO - Producing neural crest cells from human induced pluripotent stem cells

Insufficient production of erythropoietin (EPO) leads to anaemia. Developing methods for the generation and transplantation of EPO-producing cells would allow scientists to design personalised therapeutic solutions. Here we present a simple and highly reproducible protocol for the generation of neural crest cells (NCCs) that can produce and secrete erythropoiesis-competent EPO in response to hypoxia.


Specifications table
Subject Area:

Biochemistry, Genetics and Molecular Biology
More specific subject area: Erythropoietin -producing neural crest cells Protocol name: Differentiation of erythropoietin -producing neural crest cells from human induced pluripotent stem cells Reagents/tools: Included in each section of the protocol Experimental design: Human induced pluripotent stem cells are cultured in a chemically defined medium to differentiate into neural crest cells. Differentiated cells can produce functional erythropoietin in response to hypoxia. Trial registration: N/A Ethics: N/A ( continued on next page )

Value of the Protocol:
• This is a simple and reproducible differentiation protocol to generate neural crest cells from hiPSCs • The differentiated neural crest cells are fully competent to produce functional erythropoietin in response to hypoxia • This protocol can provide the basis for the development of a novel therapeutic cell-based approach to treat anaemia

Description of protocol
In patients who suffer from chronic kidney diseases (CKD), the renal erythropoietin (EPO)producing (REP) cells, which release EPO under hypoxic conditions and stimulate erythropoiesis, lose their capacity to produce EPO, leading to renal anaemia. The main available treatment for these patients is recombinant human erythropoietin (rhEPO) administration, which remains subject to various limitations, such as its high cost and adverse effects.
Here we report a protocol to differentiate human induced pluripotent stem cells (hiPSCs) into neural crest cells (NCCs) and show that these cells can produce functional EPO when cultured under hypoxic conditions ( Fig. 1 a), and induce erythropoiesis both in vitro and in vivo. Human iPSCs clone IV was obtained from neonatal fibroblasts using STEMCCA lentivirus. Cells were maintained in mTeSR1 complete medium and cultured under standard conditions (37 °C, 5% CO 2 , 20% O 2 ) on Growth-Factor-Reduced Basement Membrane Matrix-coated 100 × 20 mm Petri Dishes. Medium was changed daily. When 80% confluent, hiPSCs can be harvested as follows: 5. Resuspend in mTeSR1 complete medium containing 10 μM ROCK-inhibitor (2 mL/well) and seed with a concentration of 3 × 10 4 viable cells/cm 2 onto Growth-Factor-Reduced Basement Membrane Matrix-coated 6-well cell culture plate for 24 hours.

After 24 hours, remove the medium from each well and replace it with the same amount of Basal
Medium supplemented with 5 mM of fresh lithium chloride for 8 days. Change the medium daily. 7. By day 8 of differentiation, the majority of differentiated cells express NCC-specific markers such as AP2 α, SOX10 and HNK1 ( Fig. 1 b, c). For full differentiation markers analysis see the original paper [3] .
Note: 24 hours after medium change, cells are organised into small groups interconnected by cellular ramification. At day 2 of differentiation there are clusters of various sizes characterised by more flattened and elongated cells on the edges. Between days 3 and 4 cells undergo a significant amount of cell death. The first migrating cells can be observed at day 6 or 7 of differentiation ( Fig. 1 d). During differentiation, cells change morphology by replacing the keratin filaments with vimentin intermediate filaments at the level of lamellipodial regions.
-10 0 0 μL adjustable single channel micropipette (P10 0 0) with disposable sterile tips. At the end of hypoxia, remove the plates from the incubator and prepare the sample for EPO functional assay, ELISA, real time qRT-PCR and immunofluorescence analysis: 1. For the functional assay and ELISA test, collect and pool together the supernatant from two wells at a time in a microcentrifuge tube. Measure the total volume of the collected supernatant, aliquot and store at -20 °C until use. 2. Wash the cells twice with PBS 1X, detach them by adding 0.5 mL of prewarmed Trypsin-EDTA 0.05% to each well and incubate for 2 minutes at 37 °C. Count the cells from two wells at a time in order to relate the cell number to the EPO production for ELISA analysis. 3. For immunofluorescence analysis, wash the cells twice with PBS 1X and fix them in 4% PFA for 10 minutes at room temperature. Once fixed, they can be preserved in PBS 1X at 4 °C for extended periods (several months). 4. For real time qRT-PCR detach the cells by adding 0.5 mL of prewarmed Trypsin-EDTA 0.05% in each well and incubate for 2 minutes at 37 °C. Collect the cells, count them and centrifuge 200 x g for 5 minutes. Store the cell pellet at -80 °C.
Immunofluorescence analysis of differentiated NCCs showed that there was an increased EPO production in hypoxic conditions compared to normoxia ( Fig. 1 e, f). This observation was further confirmed by ELISA analysis of EPO released in NCCs supernatant ( Fig. 1 g).
To evaluate if the EPO produced was fully functional, we tested its capacity to induce in vitro differentiation of CD34 + haematopoietic stem cells (HSCs) into erythroblasts by following a clonogenic (colony formation) assay [3 , 4] . The group exposed to the EPO-containing supernatant formed significantly more erythroid colonies than the control group, confirming NCC-produced EPO functionality. Moreover, hiPSC-NCCs can be transplanted directly under the skin of anaemic mice to treat anaemia. Cell transplantation was shown to accelerate haematocrit restoration and to induce splenic erythropoiesis after anaemia. The detailed protocols of immunofluorence analysis and functional studies can be found in the original article [3] .