Stem Cell Reports, Volume 11
Supplemental Information
A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells Haishuang Lin, Qian Du, Qiang Li, Ou Wang, Zhanqi Wang, Neety Sahu, Christian Elowsky, Kan Liu, Chi Zhang, Soonkyu Chung, Bin Duan, and Yuguo Lei
Supplemental Information A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem CellsDerived Endothelial Cells Haishuang Lin1, Qian Du2, Qiang Li1,3, Ou Wang1,3, Zhanqi Wang4, Neety Sahu1, Christian Elowsky5, Kan Liu2, Chi Zhang2, Soonkyu Chung6, Bin Duan7 and Yuguo Lei1,3,7,8* 1: Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Nebraska, USA 2: Department of Biological Systems Engineering, University of Nebraska-Lincoln, Nebraska, USA 3: Biomedical Engineering Program, University of Nebraska-Lincoln, Nebraska, USA 4: Department of Vascular Surgery, Beijing Anzhen Hospital of Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing, China 5: Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Nebraska, USA 6: Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE, USA 7: Mary and Dick Holland Regenerative Medicine Program, University of Nebraska Medical Center, Omaha, Nebraska, USA 8: Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, Nebraska, USA
* Corresponding Author Yuguo Lei 820 N 16th St Lincoln, NE 68588 Email:
[email protected] Phone: 402-472-5313 Fax: 402-472-6989
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Figure S1. Starting hPSCs (H9, Fib-iPSCs and MSC-iPSCs). (A, E, I) Phase images. Scale bar, 200 µm. (B, F, J) Majority cells express the pluripotency markers: OCT3/4 and NANOG. Scale bar, 50 µm. (C, G, K) They can be differentiated into all three germ layer cells such as NESTIN+ ectodermal, α-SMA+ mesodermal and HNF-3β+ endodermal cells in EB assay. Scale bar, 50 µm. (D, H, L) They form teratomas containing all three germ layer tissues (arrows) in SCID mice. Scale bar, 100 µm. (A-D): H9 cells; (E-H): Fib-iPSCs; (I-L) MSC-iPSCs.
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Figure S2. Culture iPSCs in 3D thermoreversible hydrogels. (A, D) Phase images of day 0, 1, 3 and 5 iPSCs. Scale bar, 200 µm. (B, E) Immunostaining of day 5 iPSC spheroids for pluripotency marker OCT3/4, NANOG, ALP and SSEA4. Scale bar, 50 µm. (C, F) Live/dead staining of harvested day 5 iPSC spheroids. Scale bar, 200 µm.
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Figure S3. Differentiate hPSCs (including H9 hESCs and Fib-iPSCs) into ECs in 2D cultures. (A) Illustration of the differentiation protocol. (B, H) Phase images of day 0, 3 and 5 cells, respectively. Scale bar, 50 µm. (C, D, I, J) Immunostaining and flow cytometry analysis of EC markers PECAM1 (CD31) and VE-Cadherin (CD144) on day 5 cells. Scale bar, 50 µm. (E-G, K, L) The produced cells contain a small fraction of SM22A+ cells (E, K) and CD140b+ cells (F), but no OCT3/4+/NANOG+ undifferentiated H9s (G, L). Scale bar, 50 µm.
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Figure S4. Differentiate Fib-iPSCs into ECs in 3D thermoreversible PNIPAAm-PEG hydrogels. (A) Phase images of day 0, 3 and 5 cells. Scale bar, 100 µm. (B) Live/Dead staining of harvested day 5 cells. Scale bar, 50 µm. (C-E) Immunostaining and flow cytometry analysis of EC markers PECAM1 and VE-Cadherin on day 5 cells. Scale bar, 50 µm. (F) ~ 2x107 ECs are produced per milliliter of hydrogel on day 5. Data are represented as mean ± SD of three independent replicates (n=3). (G-J) The day 5 spheroids are dissociated into single cells and plated on 2D surface overnight. Immunostaining and flow cytometry shows a small fraction of SM22A+ cells (G) and CD140b+ cells (H), respectively, but no OCT3/4+/NANOG+ undifferentiated Fib-iPSCs (I). Majority cells are PECAM1+/VE-Cadherin+ (J). Scale bar, 50 µm.
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Figure S5. Functions of ECs derived from Fib-iPSCs in 3D hydrogel (3D-ECs) and 2D culture (2D-ECs). (A) Both ECs uptake fluorescence-labelled acetylated LDL (Ac-LDL). Scale bar, 50 µm. (B, C, D) Both form tubular network when plated on Matrigel for 24 hours. The co-plated vascular smooth muscle cells (VSMCs) attach to ECs. Data are represented as mean ± SD of three independent replicates (n=3). Scale bar, 50 µm. *p
