人多能幹細胞（hPSCs）包括人胚胎幹細胞（hESCs）和人誘導多能幹細胞（hiPSCs），具有分化成內胚層、外胚層和中胚層等三個胚層的細胞的能力。 近年含異種的Matrigels被用於培養及分化 hPSC，其中Matrigels由含異種的膠原蛋白 IV、層粘連蛋白-111 等組成。此外，含異種的水凝膠的臨床應用受到限制，因為它們源自動物且化學成分不確定。 根據先前的研究單一種細胞外基質 (ECM) 衍生的寡肽接枝水凝膠常被使用於 hPSC 培養和分化。考量到 Matrigels 中包含的多種成分，我們開發混合肽接枝水凝膠，它可以利用細胞上不同種類的整合素來改善 hPSC 增殖和分化。我們使用N-(3-二甲基氨基丙基)-N′-乙基碳二亞胺鹽酸鹽 (EDC) 和 N-羥基琥珀酰亞胺 (NHS) 化學將幾種類型的層粘連蛋白(KKGCGGKGGPMQKMRGDVFSP)和玻連蛋白衍生(GCGGKGGPQVTRGDVFTMP)的寡肽移植到 PVA-IA 水凝膠上。其中將水凝膠的彈性控制在 25.3 kPa（24 小時交聯時間）。 與單一肽接枝水凝膠相比，人 PSC 可以在混合肽接枝水凝膠上有效增殖。 用低濃度混合肽 (100:100 μg/mL) 製備的水凝膠可以很好地貼附、維持多能性及連續繼代超過10次。 此外，在寡肽的第一個序列上插入正氨基酸（K，賴氨酸）有助於提高寡肽接枝水凝膠的表面接枝密度，即 KKGCGGKGGPMQKMRGDVFSP、KGCGGKGGPQVTRGDVFTMP。 這解釋了肽第一位側鏈上的氨基（KKGCGGKGGPMQKMRGDVFSP）比肽主鏈上的氨基（GCGGKGGPMQKMRGDVFSP）更具反應性。 此外，在寡肽上插入帶正電的賴氨酸 (K) 有助於增強水凝膠的 zeta 電位，此特性有利於細胞的貼附。 我們的含有賴氨酸的混合肽接枝水凝膠有望促進 hPSC 的增殖效率和分化為具有低貼附性的分化細胞，例如神經細胞和視網膜色素上皮細胞。;Human pluripotent stem cells (hPSCs) including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the ability to differentiate into the cells derived from three germ layers, such as endoderm, ectoderm and mesoderm. Xeno-containing Matrigels are typically used to culture and differentiate hPSCs where Matrigels consist of xeno-containing collagen IV, laminin-111, etc. Moreover, clinical application of the xeno-containing hydrogels is limited since they are derived from animal and not chemically defined. Typically, single extracellular matrix protein (ECM)-derived oligopeptide-grafted hydrogels have been used for hPSC culture and differentiation in xeno-free culture conditions. Considering multiple components contained in Matrigels, I designed mixed peptide-grafted hydrogels, which may improve hPSC proliferation and differentiation using different binding sites of hPSCs. I prepared vitronectin-derived peptide (GCGGKGGPQVTRGDVFTMP) and laminin β4-derived peptide (KKGCGGKGGPMQKMRGDVFSP)-grafted poly(vinylalcohol-co-itaconic) hydrogels using N-hydroxysuccinimide (NHS)/1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) chemistry where the elasticity of the hydrogels was controlled at 25.3 kPa (24h crosslinking time). Human PSCs could proliferate on the mixed peptide-grafted hydrogels compared to single peptide-grafted hydrogels efficiently. Hydrogels prepared with low concentration of mixed peptides (100:100 μg/mL) could support hPSC adhesion and pluripotency. Furthermore, positive amino acid (K, lysine) insertion on the first sequence of the peptide contributed to the enhancement of surface grafting density of peptide-grafted hydrogels, i.e., KKGCGGKGGPMQKMRGDVFSP and KGCGGKGGPQVTRGDVFTMP. This is explained that the amino group on the side chain of the first position of peptide (KKGCGGKGGPMQKMRGDVFSP) is more reactive than the amino group of the main chain of the peptide (GCGGKGGPMQKMRGDVFSP). Furthermore, insertion of positive lysine (K) on the peptide contributed to enhance zeta potential of the hydrogels, which is favor to adhere hPSCs. Our mixed peptide-grafted hydrogels containing lysine and joint peptide are expected to promote efficient proliferation and differentiation of hPSCs into differentiated cells with low adhesion such as neural cells and retinal pigment epithelium cells.
