Background and Objective

• This patent describes methods and compositions for modulating the electrical potential across cell membranes to influence cell behavior.
• The approach uses naturally occurring (endogenous) ligand‐gated ion channels to adjust the cell’s membrane voltage.
• The primary goals are to promote tissue regeneration, control cell proliferation and differentiation, and even inhibit unwanted cell growth such as cancer.

Key Concepts and Terminology

• Membrane Potential: The voltage difference between the inside and outside of a cell that influences cellular functions.
• Ligand-Gated Channels: Protein channels that open or close in response to specific chemical signals (ligands).
• Macrocyclic Lactones: A class of compounds (e.g., ivermectin, avermectin) that can open these channels and alter membrane potential.
• Instructor Cells: Specific cells that, when their membrane potential is modulated, non-cell-autonomously influence the behavior of neighboring (responder) cells.
• Progenitor Cells: Cells with the capacity to proliferate and differentiate into specialized cell types, similar to stem cells.

Method Overview (Step-by-Step)

• Step 1: Select a macrocyclic lactone (for example, ivermectin) that acts on endogenous ligand-gated channels.
• Step 2: Apply the compound to cell cultures or embryos to alter the membrane potential.
• Step 3: Adjust the extracellular ionic environment (e.g., by changing chloride ion concentration) to control whether cells become depolarized (less negative) or hyperpolarized (more negative).
• Step 4: Monitor the changes in cell behavior – such as increased proliferation, altered cell shape, and migration patterns.
• Step 5: Identify instructor cells by detecting the expression of specific ion channels (e.g., the GlyCl channel) that mediate these effects.
• Step 6: Use observable outcomes, like hyperpigmentation in Xenopus embryos, as a measurable sign of successful membrane modulation.
• Step 7: Explore therapeutic applications by tailoring the modulation method to either promote tissue regeneration or inhibit unwanted cell proliferation (as in cancer treatment).

Experimental Findings and Examples

• Example 1: Treatment of Xenopus embryos with ivermectin led to hyperpigmentation—an outcome linked to increased proliferation and migration of pigment (melanocyte) cells.
• Example 2: Early exposure to ivermectin (during gastrulation and neurulation) significantly increased the number of melanocytes, whereas later exposure only changed cell shape.
• Example 3: Varying extracellular chloride levels confirmed that membrane depolarization is key to triggering the observed cellular effects.
• Example 4: The addition of fluoxetine (a selective serotonin reuptake inhibitor) blocked ivermectin-induced hyperpigmentation, suggesting that the serotonin pathway plays a role in downstream signaling.
• Example 5: In human melanocyte cultures, increasing extracellular potassium (using potassium gluconate) induced a similar cell shape change, indicating that depolarization affects cell morphology.
• Example 6: Blocking the GlyCl channel with strychnine produced alternative effects (such as expansion of the cement gland), highlighting the specificity of different ion channels in regulating cell fate.

Applications and Therapeutic Implications

• These methods can promote tissue regeneration by inducing controlled cell proliferation and differentiation.
• They offer potential in cancer treatment by inhibiting proliferation in cells that are abnormally depolarized.
• The approach allows for non-invasive control of cell behavior using small molecules that modulate endogenous ion channels.
• The techniques provide a novel screening method for candidate therapeutic agents based on their ability to alter membrane potential.

Additional Technical Details

• The patent describes multiple embodiments that use various ligand-gated channels (such as chloride and potassium channels) to fine-tune cell behavior.
• Methods include both direct modulation of target cells and indirect modulation via instructor cells that influence other (responder) cells.
• Precise control over depolarization or hyperpolarization is achieved by adjusting the extracellular ion concentrations.
• Extensive experimental protocols (e.g., in situ hybridization, microinjections, voltage imaging) validate the effectiveness of these approaches.

Summary of Key Conclusions

• Modulating the membrane potential is an effective way to control cellular behavior.
• Macrocyclic lactones like ivermectin can selectively activate endogenous ion channels to induce desired changes in cells.
• Instructor cells play a crucial role in non-cell-autonomous regulation of cell fate.
• This approach has wide-ranging applications in regenerative medicine and cancer therapy.

Future Directions

• Further research may explore additional ion channel modulators and their combinations for more effective therapies.
• Screening for candidate compounds using membrane potential modulation could accelerate the discovery of new regenerative or anti-cancer treatments.

背景和目的

• 本专利介绍了一种调节细胞膜电位以影响细胞行为的方法和组合物。
• 该方法利用细胞内天然存在的配体门控离子通道来调整细胞膜的电压。
• 主要目标是促进组织再生，控制细胞增殖与分化，并抑制不希望出现的细胞过度生长（如癌症）。

关键概念和术语

• 膜电位：细胞内外的电压差，对细胞功能具有重要影响。
• 配体门控通道：当特定化学信号（配体）出现时，细胞膜上的蛋白质通道会打开或关闭。
• 大环内酯类：一类化合物（如伊维菌素、阿维菌素），可激活这些通道并改变膜电位。
• 指令细胞：当其膜电位被调控后，可以非细胞自主地影响邻近响应细胞行为的特定细胞。
• 祖细胞：具有增殖和分化为更专门细胞能力的细胞，类似于干细胞。

方法概述（步骤解析）

• 步骤1：选择一种大环内酯类化合物（例如伊维菌素），通过激活内源性配体门控通道来调节膜电位。
• 步骤2：将该化合物应用于细胞培养或胚胎中，以改变细胞膜的电状态。
• 步骤3：调节细胞外离子环境（例如改变氯离子浓度），以控制细胞是去极化（变得较少负）还是超极化（变得更负）。
• 步骤4：监测细胞行为变化，如细胞增殖增加、细胞形态改变和迁移模式变化。
• 步骤5：通过检测特定离子通道（如GlyCl通道）的表达，确定“指令细胞”，这些细胞对调控效果至关重要。
• 步骤6：利用例如非洲爪蟾胚胎中出现的过度色素沉着作为调控成功的可观察指标。
• 步骤7：探索该方法在组织再生和抑制癌细胞过度增殖等治疗中的应用潜力。

实验发现和实例

• 实例1：利用伊维菌素处理非洲爪蟾胚胎，导致色素细胞增多和迁移，产生过度色素沉着现象。
• 实例2：在胚胎早期（胚泡形成和神经管形成期）暴露于伊维菌素显著增加了黑色素细胞数量，而晚期暴露仅改变了细胞形态。
• 实例3：通过调节细胞外氯离子浓度，证明膜去极化是触发这些细胞效应的关键因素。
• 实例4：使用氟西汀（一种选择性血清素再摄取抑制剂）阻断下游信号后，抑制了伊维菌素引起的过度色素沉着，表明血清素信号通路的作用。
• 实例5：在人体黑色素细胞培养中，通过提高外部钾离子浓度诱导细胞去极化，导致细胞形态变化，与非洲爪蟾的情况相似。
• 实例6：使用士的宁阻断GlyCl通道，改变了水泥腺的发育，显示出不同离子通道调控在细胞命运调控中的特异性。

应用和治疗意义

• 该方法可通过诱导细胞增殖和分化来促进组织再生。
• 它在癌症治疗中具有潜力，通过抑制异常去极化细胞的过度增殖来控制癌症。
• 此技术允许利用小分子和特定离子通道调控剂以非侵入性方式调节细胞行为。
• 该方法还提供了一种新型筛选候选治疗剂的手段，依据其改变膜电位的能力。

其他技术细节

• 专利描述了利用不同类型的配体门控通道（如氯离子通道、钾通道等）来调控细胞行为的多个实施例。
• 方法既包括直接调控目标细胞，也包括通过指令细胞间接调控响应细胞的行为。
• 通过调节细胞外离子浓度，可以精确控制去极化或超极化的程度。
• 大量实验方案（如原位杂交、微注射、电压成像等）验证了这些方法的有效性。

关键结论总结

• 调节膜电位是一种有效控制细胞行为的方法。
• 利用如伊维菌素之类的大环内酯类化合物可以选择性激活内源性离子通道，从而诱导预期的细胞反应。
• 指令细胞在非细胞自主调控中发挥着关键作用，能影响邻近细胞的增殖与分化。
• 该方法在再生医学和癌症治疗领域展现出广泛的应用前景。

未来研究方向

• 未来研究可探索更多种类的离子通道调控剂及其组合治疗的潜力。
• 利用该方法筛选候选化合物，有望加速新型再生或抗癌治疗剂的发现。