Department of Biomedical Engineering, The University of Texas at Austin, 107 W Dean Keeton St., Austin, TX, 78712-1139, USA.
Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA.
Stem Cell Res Ther. 2022 Mar 28;13(1):131. doi: 10.1186/s13287-022-02801-7.
Spinal interneurons (INs) relay sensory and motor control information between the brain and body. When this relay circuitry is disrupted from injury or disease, it is devastating to patients due to the lack of native recovery in central nervous system (CNS) tissues. Obtaining a purified population of INs is necessary to better understand their role in normal function and as potential therapies in CNS. The ventral V0 (V0) INs are excitatory neurons involved in locomotor circuits and are thus of interest for understanding normal and pathological spinal cord function. To achieve scalable amounts of V0 INs, they can be derived from pluripotent sources, such as mouse embryonic stem cells (mESCs), but the resultant culture is heterogenous, obscuring the specific role of V0 INs. This study generated a transgenic mESC line to enrich V0 INs from induced cultures to allow for a scalable, enriched population for future in vitro and in vivo studies.
The transgenic Evx1-PAC mESC line was created by CRISPR-Cas9-mediated insertion of puromycin-N-acetyltransferase (PAC) into the locus of V0 IN marker Evx1. Evx1 and PAC mRNA expression were measured by qPCR. Viability staining helped establish the selection protocol for V0 INs derived from Evx1-PAC mESCs inductions. Immunostaining was used to examine composition of selected inductions. Cultures were maintained up to 30 days to examine maturation by expression of mature/synaptic markers, determined by immunostaining, and functional activity in co-cultures with selected motor neurons (MNs) and V2a INs on microelectrode arrays (MEAs).
V0 IN inductions were best selected with 4 µg/mL puromycin on day 10 to 11 and showed reduction of other IN populations and elimination of proliferative cells. Long-term selected cultures were highly neuronal, expressing neuronal nuclear marker NeuN, dendritic marker MAP2, pre-synaptic marker Bassoon, and glutamatergic marker VGLUT2, with some cholinergic VAChT-expressing cells. Functional studies on MEAs showed that co-cultures with MNs or MNs plus V2a INs created neuronal networks with synchronized bursting.
Evx1-PAC mESCs can be used to purify V0 IN cultures for largely glutamatergic neurons that can be used in network formation studies or for rodent models requiring transplanted V0 INs.
脊髓中间神经元（INs）在大脑和身体之间传递感觉和运动控制信息。当这种中继电路因损伤或疾病而中断时，由于中枢神经系统（CNS）组织中缺乏天然的恢复，对患者来说是毁灭性的。获得纯化的 INs 群体对于更好地了解它们在正常功能中的作用以及作为 CNS 的潜在治疗方法是必要的。腹侧 V0（V0）INs 是参与运动回路的兴奋性神经元，因此对于理解正常和病理性脊髓功能很有意义。为了获得大量的 V0 INs，可以从多能性来源（如小鼠胚胎干细胞（mESCs））中获得，但所得培养物是异质的，掩盖了 V0 INs 的特定作用。本研究生成了一种转基因 mESC 系，通过 CRISPR-Cas9 介导将嘌呤霉素-N-乙酰转移酶（PAC）插入 V0 IN 标记物 Evx1 的基因座中，从诱导培养物中富集 V0 INs，从而获得可用于未来体外和体内研究的可扩展、富集的 V0 INs 群体。
通过 CRISPR-Cas9 介导将嘌呤霉素-N-乙酰转移酶（PAC）插入 V0 IN 标记物 Evx1 的基因座中，创建了转基因 Evx1-PAC mESC 系。通过 qPCR 测量 Evx1 和 PAC mRNA 的表达。通过活力染色来建立从 Evx1-PAC mESCs 诱导物中分离 V0 IN 的选择方案。免疫染色用于检查所选诱导物的组成。培养物维持长达 30 天，通过免疫染色检测成熟的/突触标记物的表达来检查成熟度，并在微电极阵列（MEA）上与选定的运动神经元（MNs）和 V2a INs 共培养来检测功能活性。
V0 IN 诱导物在第 10 天至 11 天用 4μg/mL 嘌呤霉素进行最佳选择，并且减少了其他 IN 群体并消除了增殖细胞。长期选择的培养物高度神经元化，表达神经元核标记物 NeuN、树突标记物 MAP2、前突触标记物 Bassoon 和谷氨酸能标记物 VGLUT2，并有一些表达胆碱能 VAChT 的细胞。MEA 上的功能研究表明，与 MNs 或 MNs 加 V2a INs 共培养可以形成具有同步爆发的神经元网络。
Evx1-PAC mESCs 可用于纯化 V0 IN 培养物，用于主要为谷氨酸能神经元的网络形成研究，或用于需要移植 V0 IN 的啮齿动物模型。
