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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), <t>SLC4A11,</t> N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.
Slc4a11, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), <t>SLC4A11,</t> N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.
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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), <t>SLC4A11,</t> N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.
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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), <t>SLC4A11,</t> N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.
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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), <t>SLC4A11,</t> N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.
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269356 slc4a11 b6n va  (Cyagen Biosciences)
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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), <t>SLC4A11,</t> N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.
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Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), SLC4A11, N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.

Journal: MedComm

Article Title: High‐Purity Functional Corneal Endothelial Cells From Human Induced Pluripotent Stem Cells via a Novel Wash‐Out Method

doi: 10.1002/mco2.70650

Figure Lengend Snippet: Characterization of corneal endothelial‐like cells (CECs) generated from induced pluripotent stem cells (iPSCs). (A) The wash‐out protocol enables more differentiated CECs to adhere at a faster rate to the VTN‐coated surface during the initial stage, while relatively immature cells with weaker attachment capacity are subsequently washed out and removed. A control culture dish without the wash‐out step is used for comparison of the initial cell seeding number. When approximately 50% of the seeded cells had attached to the culture surface, the remaining unattached cells were removed by aspirating the medium containing unattached cells, followed by a single gentle wash with PBS for less than 1 min. The culture was then replenished with fresh medium at the same volume routinely used for maintenance and returned to incubation. The wash‐out protocol was applied to the ACE2 cell population at each passage during the differentiation process and was performed once weekly upon reaching confluence, resulting in two wash‐out procedures during the 14‐day differentiation protocol and three during the 21‐day protocol. (B) Protocol for the differentiation of iPSCs into CECs. iPSCs were differentiated into CECs through either a neural crest cell (NCC) intermediate stage or directly, without the NCC stage. Phase‐contrast microscopy revealed CEC‐specific hexagonal morphology after the differentiation. Scale bars = 100 µm. (C) Immunocytochemistry for OCT4 and NANOG, iPSC markers, and CD166, a CEC marker, on iPSC colonies. No CD166‐IR was detected in the iPSC colonies. Scale bars = 50 µm (OCT4, CD166); scale bars = 20 µm (NANOG). (D) Immunocytochemistry for a group of CEC‐specific markers; zona occludens‐1 (ZO‐1; a tight‐junction protein), pump function protein Na + /K + ATPase α1 (ATP1A1), SLC4A11, N‐CADHERIN, CD166, and NCAM on iPSC‐derived CECs. Scale bars = 100 µm.

Article Snippet: The antibodies used included human anti‐ZO‐1 (1:250; Life Technologies), SLC4A11 (1:250; Novus, Centennial, CO), N‐CADHERIN (1:200; Cell Signaling Technology, Danvers, MA), Na + /K + ATPase α1 (1:200; Santa Cruz Biotechnology, Dallas, TX), CD166 (1:500; BD Biosciences, Franklin Lakes, NJ), PITX2 (100 μg/mL; Abnova, Taipei), and NCAM (1:200; R&D Systems, Minneapolis, MN).

Techniques: Generated, Control, Comparison, Gentle, Incubation, Microscopy, Immunocytochemistry, Marker, Derivative Assay