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小分子化合物在msc细胞培养中的研究进展
小分子化合物在间充质干细胞 (MSC) 培养中的应用研究取得了显著进展，主要体现在以下几个方面：促进MSC增殖、维持干性、调控分化、改善治疗效果以及在3D培养系统中的应用。这些小分子化合物通过影响MSC的微环境、细胞内信号通路和基因表达，从而优化MSC的体外培养条件，克服MSC在临床转化中面临的一些挑战。
1. 维持MSC的干性和促进增殖
MSC在体外大规模扩增过程中常常会失去其再生特性和干性，这严重阻碍了其临床应用。小分子化合物被广泛研究以解决这一问题：
- 改善MSC的再生特性和分泌功能： 水凝胶培养平台可以通过调节MSC微环境来促进MSC增殖并维持其再生特性（如干性和分泌功能）。在此类系统中，生化信号（如小分子、肽和蛋白质）可以改善并指导MSC的分泌谱，从而提高临床转化效果。
- 延缓衰老并保持干性：
- 褪黑素（Melatonin）： 褪黑素被发现能有效保留长期传代（15代）的骨髓MSC（BMMSC）的自我更新和分化能力。它通过激活抗氧化防御系统、抑制细胞衰老途径和保留干性相关基因的表达来发挥作用，从而改善了长期培养BMMSC在骨再生和免疫治疗中的体内治疗效果。
- 白藜芦醇（Resveratrol, RSV）： RSV被证明可以维持长期培养（从原代培养开始6周）MSC的增殖和分化潜力，即使达到有效细胞剂量（ECD）也能保持。RSV的作用机制依赖于MSC培养中的SIRT1-SOX2轴，并且在体内大鼠颅骨缺损模型中显著改善了MSC移植后的骨再生。
- 整合应激反应（ISR）的激活： 三维（3D）聚集培养可以逆转人MSC在2D硬塑料培养中出现的异常增殖、代谢转变和自噬活性降低等问题。研究表明，3D聚集培养通过提高自噬和恢复代谢状态至原始表型，显著增加了骨髓MSC（bMSC）和脂肪组织MSC（ASC）的缺血存活率，这与激活真核起始因子2（eIF2）通路和整合应激反应（ISR）有关。用小分子化合物salubrinal和GSK2606414扰动ISR对bMSC、ASC和成纤维细胞（FB）产生了不同的反应。这表明基于聚集的预处理培养通过建立ISR和稳态有可能提高扩增人MSC的治疗效果。
- 物理、化学和生化刺激的影响： 除了小分子，物理刺激（如刚度、几何形状和拓扑结构）、化学刺激（如改变表面化学）以及生化刺激（如细胞因子、激素、小分子和草药提取物）都被研究用于促进人MSC（h-MSC）的高产量生产，同时不丧失功能特性。然而，这些策略尚未广泛应用于工业化生产。
2. 调控MSC的分化
小分子化合物在引导MSC向特定细胞谱系分化方面展现出巨大潜力，这对于细胞疗法和组织工程至关重要：
- 骨骼和软骨分化：
- 表观遗传调节分子： 研究筛选了84种小分子药理学试剂库，发现其中一些可以调节核小体修饰，从而影响人MSC（hMSCs）的成骨分化。例如，吉西他滨（Gemcitabine）和氯米他宾（Chidamide）可以显著促进老年供体来源hMSCs的成骨分化，分别提高5.9倍和2.3倍。相反，其他分子（如4-Iodo-SAHA、Scriptaid、AGK2、CI-amidine和Delphidine Chloride）能最大程度地抑制成骨分化。这表明小分子表观遗传分子可以通过调节细胞的表观遗传谱来敏感地调控骨髓来源MSC的谱系可塑性命运。
- Kartogenin（KGN）纳米颗粒： KGN是一种促进软骨分化的小分子药物。将其负载到3D打印的重组胶原/壳聚糖甲基丙烯酸酯/纳米粘土水凝胶中，可以实现KGN的持续释放，从而诱导人骨髓MSC（hBMSCs）分化为软骨细胞，显著增强胶原II和糖胺聚糖的表达。体内研究也表明KGN负载支架能促进软骨组织再生。
- Ruxolitinib（INCB018424）： 作为JAK-STAT信号通路的抑制剂，ruxolitinib（3 μM）被发现能抑制hMSCs的成骨分化和体外基质矿化，并减少体内的异位骨形成。这提示ruxolitinib可能为治疗加速成骨分化和矿化引起的病理状况提供新的治疗策略。
- 工程化形态发生场： 通过在聚合物支架系统中结合时空控制的形态发生因子，可以创建精确的形态发生场来指导MSC的多谱系分化。例如，结合特定抑制剂（中和抗体或小分子激酶抑制剂）与形态发生因子（如成牙本质的TGF-β1和成骨的BMP4），可以设计出具有不同空间隔离信号区域的支架，从而诱导MSC在特定区域进行矿化组织分化。
- 胰岛素生产细胞分化： 2型糖尿病的细胞疗法研究正在利用干细胞分化为胰岛素生产细胞（IPCs），包括胚胎干细胞（ESCs）、MSC、诱导多能干细胞（iPSCs）以及来自成人胰腺、肝脏、中枢神经系统、骨髓和脂肪组织的其他干细胞。成功诱导iPSCs的关键在于可用干细胞的数量和质量，以及适应性方案的开发，这些方案要能确定外部因子和小分子参与的环境。
- 神经元和神经嵴细胞分化：
- 多巴胺能神经元分化： 星形胶质细胞（AS）和MSC被认为是诱导多巴胺能（DA）神经元的有前途的候选细胞。研究发现，AS可以被转录因子混合物Mash1、Lmx1a和Nurr1（MLN）、化学混合物（S/C/D）和形态发生因子SHH、FGF8和FGF2（S/F8/F2）有效诱导为DA神经元。相比之下，来自脐带（UC-MSCs）或脂肪组织（AD-MSCs）的MSC在S/F8/F2诱导下表现出中等程度的TH免疫反应性，而非MLN或S/C/D。这表明AS和MSC在诱导为DA神经元方面具有谱系特异性分子编码，并突显了AS在帕金森病（PD）细胞替代疗法中的独特优势。
- 神经嵴谱系细胞： 通过两步小分子化合物混合物处理6天，可以从人脂肪来源MSC（ADSCs）中直接诱导神经嵴谱系细胞（NCCs）。这种方法显著上调了神经嵴标志物（如NGFR和SOX10）的mRNA和蛋白表达，以及各种营养因子。还识别出CD271和CD57等神经嵴特异性细胞表面标志物被明显上调。这为研究人NCCs在细胞疗法和再生医学中的应用提供了一个有前景的实验平台。
- 肾上皮样细胞分化： 人脐带来源MSC（hucMSCs）可以分化成肾上皮样细胞。通过优化视黄酸、活化素-A和骨形态发生蛋白-7（BMP-7）的组合浓度，以及Wnt信号通路激活剂（CHIR99021）的预处理，hucMSCs显示出明显的上皮细胞样分化，包括表型变化以及基因和蛋白质表达的上调，与上皮细胞表型一致。
- 脂肪细胞分化： 增强骨髓MSC（BM-MSCs）的脂肪细胞分化可能导致再生障碍性贫血（AA）骨髓微环境的脂肪堆积改变。寻找抑制BM-MSCs脂肪生成的药物可能是AA治疗的新方向。研究发现，大黄素（emodin）能显著抑制AA BM-MSCs的体外脂肪生成分化。其机制涉及emodin增加Tribbles同源物3（TRIB3）的表达，TRIB3在AA BM-MSCs中表达显著降低，且被证明是AA BM-MSCs脂肪生成的负调节因子。emodin介导的TRIB3上调减轻了AA BM-MSCs的脂肪生成能力，提示emodin可能作为AA治疗的潜在方案。
- 诱导多能干细胞（iPSCs）向MSC分化： 利用小分子抑制剂可以实现人胚胎干细胞（ESCs）和iPSCs的间充质分化。通过在无血清培养基中培养ESCs/iPSCs，并添加TGF-β通路抑制剂SB431542，可以生成上皮样单层细胞。随后，将细胞转移到常规MSC培养基中完成分化。这种方法无需形成胚胎体或饲养层细胞共培养，提供了一种快速、均匀、临床适用且高效的从人iPSCs生成MSC的系统。
3. 优化MSC培养环境与递送系统
小分子化合物也用于优化MSC的培养环境和递送系统，以提高其在体内的存活率、保留率和植入成功率：
- 水凝胶平台：
- 调节MSC微环境： 水凝胶系统可以被设计用于促进MSC增殖，维持其干性和分泌功能，并改善MSC在体内的存活、保留和植入。通过定制生化和生物物理线索，可以指导MSC的分泌谱。水凝胶材料的特性（如基质模量、粘弹性、维度、细胞粘附和孔隙率）以及生化线索（小分子、肽和蛋白质）都影响MSC的分泌，并通过细胞-基质和细胞-细胞相互作用介导。
- 可调谐水凝胶： 基于生物惰性聚乙二醇（PEG）并连接特定整合素结合小分子的细胞可降解水凝胶被用于MSC递送。这些3D培养系统改变了MSC的转录组，例如，在3D水凝胶培养中，成骨分化相关基因上调，通过在水凝胶中连接整合素结合RGD肽可以部分抑制这些基因的表达。这种RGD连接的水凝胶在离体人伤口中应用时，能够支持伤口再上皮化，可能通过增加PDGF表达和减少IL-6表达来实现。
- 光降解水凝胶： 光降解水凝胶是用于细胞功能研究、组织工程和细胞递送的有用平台，因为其物理和化学性质可以通过光动态控制。通过使用基于环状亚苄基酮的小分子作为光敏剂，可以显著提高光降解水凝胶的两光子降解效率，同时保持细胞相容性。
- 3D细胞组装中的营养物质递送： 在多细胞球体等3D细胞组装中，营养物质（如氧气和葡萄糖）的扩散不足常常导致坏死核心的形成。纳米功能化微粒被开发出来作为合成中心，用于调节小分子在人MSC（hMSC）球体内的扩散。将葡萄糖负载到介孔二氧化硅纳米颗粒（MSNs）中，并将其涂覆在聚乳酸-乙醇酸共聚物（PLGA）微粒表面，可以实现葡萄糖在hMSC球体内的局部释放，从而提高球体在短期缺氧培养中的细胞活力。这种纳米功能化微粒递送系统为3D细胞组装内小分子的局部递送提供了通用平台。
- 促进造血干细胞（HSC）体外扩增： 造血干细胞的体外扩增对临床应用有多方面益处，包括改善患者生存率、将脐带血干细胞用于成人应用以及在基因改造后选择性扩增干细胞群。除了基质/HSC共培养、连续灌注和补料分批培养，补充外源配体、膜可转运转录因子、补体成分、蛋白质修饰酶、代谢物或小分子化合物也被用于HSC扩增系统。其中一些扩增系统已进入临床试验。
- 抑制肿瘤生长： 研究发现，人骨髓MSC（hBMSCs）来源的外泌体通过激活Hedgehog信号通路促进骨肉瘤（MG63）和胃癌（SGC7901）细胞的生长。通过使用Hedgehog通路的小分子抑制剂，可以显著抑制hBMSC来源外泌体对肿瘤生长的促进作用。这揭示了Hedgehog信号通路在hBMSCs来源外泌体诱导的肿瘤进展中的新作用。
总结与展望
小分子化合物在MSC培养中的研究进展为克服MSC临床应用中的诸多挑战提供了有前景的策略。它们不仅能够有效维持MSC的干性和促进增殖，还能精准调控MSC向特定谱系分化，甚至改善MSC在复杂3D培养环境和体内递送系统中的性能。然而，目前许多研究仍处于实验室阶段，要实现工业化生产和广泛的临床应用，仍需进一步研究，包括：
- 优化小分子化合物的组合和浓度： 不同的MSC来源和应用场景可能需要不同的小分子组合和浓度，以实现最佳效果。
- 深入理解作用机制： 尽管许多小分子化合物的功能已被揭示，但其在MSC细胞命运决定中的详细分子机制仍需更深入的研究，以便设计出更靶向、更有效的小分子。
- 大规模生产的可行性： 验证这些新的细胞疗法必须建立在实验依据之上，并考虑如何实现高效、低成本的MSC收集、处理和扩增系统，以满足临床需求。
- 安全性和生物相容性： 在临床转化前，需要对小分子化合物及其诱导的MSC在体内的长期安全性和生物相容性进行充分评估。
- 结合多学科方法： 未来工作将需要结合生物物理学、生物化学和材料科学等多个学科的知识，以更好地理解微环境线索如何影响MSC分泌组，并设计出具有优化生物物理和生化线索的新一代生物材料，从而指导MSC分泌谱以改善临床转化结果。
通过持续的研究和技术创新，小分子化合物有望在未来MSC培养和细胞疗法领域发挥越来越重要的作用，推动再生医学的发展。
References
1Engineering the MSC Secretome: A Hydrogel Focused Approach.PubMed
Marissa E Wechsler, Varsha V Rao, Alexandra N Borelli, et al.
The therapeutic benefits of exogenously delivered mesenchymal stromal/stem cells (MSCs) have been largely attributed to their secretory properties. However, clinical translation of MSC-based therapies is hindered due to loss of MSC regenerative properties during large-scale expansion and low survival/retention post-delivery. These limitations might be overcome by designing hydrogel culture platforms to modulate the MSC microenvironment. Hydrogel systems could be engineered to i) promote MSC proliferation and maintain regenerative properties (i.e., stemness and secretion) during ex vivo expansion, ii) improve MSC survival, retention, and engraftment in vivo, and/or iii) direct the MSC secretory profile using tailored biochemical and biophysical cues. Herein, it is reviewed how hydrogel material properties (i.e., matrix modulus, viscoelasticity, dimensionality, cell adhesion, and porosity) influence MSC secretion, mediated through cell-matrix and cell-cell interactions. In addition, it is highlighted how biochemical cues (i.e., small molecules, peptides, and proteins) can improve and direct the MSC secretory profile. Last, the authors' perspective is provided on future work toward the understanding of how microenvironmental cues influence the MSC secretome, and designing the next generation of biomaterials, with optimized biophysical and biochemical cues, to direct the MSC secretory profile for improved clinical translation outcomes.
2Small molecules and extrinsic factors promoting differentiation of stem cells into insulin-producing cells.PubMed
Gui Pan, Jianping Liu
In the modern world, type-2 diabetes mellitus has become a leading public healthcare problem, due to major risks of morbidity and mortality. Prevalence has increased significantly in recent decades. Treatment involves oral hypoglycemic agents or insulin replacement therapy. Development is ongoing for cell-based diabetes therapies using stem cells with the potential to differentiate into insulin-producing cells (IPCs): embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and stem cells from adult pancreas, liver, central nervous system, bone marrow and adipose tissue. Successful induction of iPSCs, however, depends on the quantity and quality of available stem cells and the development of adapted protocols determining the environment of extrinsic factors and involvement of small molecules. Validating such new cell therapies must be founded on this experimental rationale.
3Ex vivo expansion of hematopoietic stem cells.PubMed
JingJing Xie, ChengCheng Zhang
Ex vivo expansion of hematopoietic stem cells (HSCs) would benefit clinical applications in several aspects, to improve patient survival, utilize cord blood stem cells for adult applications, and selectively propagate stem cell populations after genetic manipulation. In this review we summarize and discuss recent advances in the culture systems of mouse and human HSCs, which include stroma/HSC co-culture, continuous perfusion and fed-batch cultures, and those supplemented with extrinsic ligands, membrane transportable transcription factors, complement components, protein modification enzymes, metabolites, or small molecule chemicals. Some of the expansion systems have been tested in clinical trials. The optimal condition for ex vivo expansion of the primitive and functional human HSCs is still under development. An improved understanding of the mechanisms for HSC cell fate determination and the HSC culture characteristics will guide development of new strategies to overcome difficulties. In the future, development of a combination treatment regimen with agents that enhance self-renewal, block differentiation, and improve homing will be critical. Methods to enhance yields and lower cost during collection and processing should be employed. The employment of an efficient system for ex vivo expansion of HSCs will facilitate the further development of novel strategies for cell and gene therapies including genome editing.
4Effects of Physical, Chemical, and Biological Stimulus on h-MSC Expansion and Their Functional Characteristics.PubMed
David A Castilla-Casadiego, Ana M Reyes-Ramos, Maribella Domenech, et al.
Human adult mesenchymal stem or stromal cells (h-MSC) therapy has gained considerable attention due to the potential to treat or cure diseases given their immunosuppressive properties and tissue regeneration capabilities. Researchers have explored diverse strategies to promote high h-MSC production without losing functional characteristics or properties. Physical stimulus including stiffness, geometry, and topography, chemical stimulus, like varying the surface chemistry, and biochemical stimuli such as cytokines, hormones, small molecules, and herbal extracts have been studied but have yet to be translated to industrial manufacturing practice. In this review, we describe the role of those stimuli on h-MSC manufacturing, and how these stimuli positively promote h-MSC properties, impacting the cell manufacturing field for cell-based therapies. In addition, we discuss other process considerations such as bioreactor design, good manufacturing practice, and the importance of the cell donor and ethics factors for manufacturing potent h-MSC.
5Aggregation-induced integrated stress response rejuvenates culture-expanded human mesenchymal stem cells.PubMed
Brent M Bijonowski, Qin Fu, Xuegang Yuan, et al.
Protein homeostasis is critical for cellular function, as loss of homeostasis is attributed to aging and the accumulation of unwanted proteins. Human mesenchymal stem cells (MSCs) have shown promising therapeutic potential due to their impressive abilities to secrete inflammatory modulators, angiogenic, and regenerative cytokines. However, there exists the problem of human MSC expansion with compromised therapeutic quality. Duringin vitro expansion, human MSCs are plated on stiff plastics and undergo culture adaptation, which results in aberrant proliferation, shifts in metabolism, and decreased autophagic activity. It has previously been shown that three-dimensional (3D) aggregation can reverse some of these alterations by heightening autophagy and recovering the metabolic state back to a naïve phenotype. To further understand the proteostasis in human MSC culture, this study investigated the effects of 3D aggregation on the human MSC proteome to determine the specific pathways altered by aggregation. The 3D aggregates and 2D cultures of human MSCs derived from bone marrow (bMSC) and adipose tissue (ASC) were analyzed along with differentiated human dermal fibroblasts (FB). The proteomics analysis showed the elevated eukaryotic initiation factor 2 pathway and the upregulated activity of the integrated stress response (ISR) in 3D aggregates. Specific protein quantification further determined that bMSC and ASC responded to ISR, while FB did not. 3D aggregation significantly increased the ischemic survival of bMSCs and ASCs. Perturbation of ISR with small molecules salubrinal and GSK2606414 resulted in differential responses of bMSC, ASC, and FB. This study indicates that aggregation-based preconditioning culture holds the potential for improving the therapeutic efficacy of expanded human MSCs via the establishment of ISR and homeostasis.
6Engineering Lineage Potency and Plasticity of Stem Cells using Epigenetic Molecules.PubMed
Anandika Dhaliwal, Sandra Pelka, David S Gray, et al.
Stem cells are considered as a multipotent regenerative source for diseased and dysfunctional tissues. Despite the promise of stem cells, the inherent capacity of stem cells to convert to tissue-specific lineages can present a major challenge to the use of stem cells for regenerative medicine. We hypothesized that epigenetic regulating molecules can modulate the stem cell's developmental program, and thus potentially overcome the limited lineage differentiation that human stem cells exhibit based on the source and processing of stem cells. In this study, we screened a library of 84 small molecule pharmacological agents indicated in nucleosomal modification and identified a sub-set of specific molecules that influenced osteogenesis in human mesenchymal stem cells (hMSCs) while maintaining cell viability in-vitro. Pre-treatment with five candidate hits, Gemcitabine, Decitabine, I-CBP112, Chidamide, and SIRT1/2 inhibitor IV, maximally enhanced osteogenesis in-vitro. In contrast, five distinct molecules, 4-Iodo-SAHA, Scriptaid, AGK2, CI-amidine and Delphidine Chloride maximally inhibited osteogenesis. We then tested the role of these molecules on hMSCs derived from aged human donors and report that small epigenetic molecules, namely Gemcitabine and Chidamide, can significantly promote osteogenic differentiation by 5.9- and 2.3-fold, respectively. Taken together, this study demonstrates new applications of identified small molecule drugs for sensitively regulating the lineage plasticity fates of bone-marrow derived mesenchymal stem cells through modulating the epigenetic profile of the cells.
7Enhancement of Mesenchymal Stem Cell-Driven Bone Regeneration by Resveratrol-Mediated SOX2 Regulation.PubMed
Yoorim Choi, Dong Suk Yoon, Kyoung-Mi Lee, et al.
Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine. However, MSCs age rapidly during long-term culture and lose their therapeutic potential before they reach effective cell doses (ECD) for cell therapy. Thus, a prerequisite for effective MSC therapy is the development of cell culture methods to preserve the therapeutic potential during long-term cultivation. Resveratrol (RSV) has been highlighted as a therapeutic candidate for bone disease. Although RSV treatment has beneficial effects on bone-forming cells, studies are lacking. The current study showed that long-term (6 weeks from primary culture date)-cultured MSCs with RSV induction retained their proliferative and differentiation potential despite reaching ECD. The mechanism of RSV action depends entirely on the SIRT1-SOX2 axis in MSC culture. In a rat calvarial defect model, RSV induction significantly improved bone regeneration after MSC transplantation. This study demonstrated an example of efficient MSC therapy for treating bone defects by providing a new strategy using the plant polyphenol RSV.
8Emodin suppresses adipogenesis of bone marrow derived mesenchymal stem cells from aplastic anemia via increasing TRIB3 expression.PubMed
Xianning Zhang, Lulu Liu, Jian Wang, et al.
BACKGROUND: Increasing evidence indicate that enhanced adipogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) could contribute to the adiposity alteration in marrow microenvironment of aplastic anemia (AA). Identifying small molecule drugs with role in inhibiting adipogenesis of BM-MSCs may represent a novel direction in AA therapy by improving BM-MSCs mediated marrow microenvironment. METHODS: For the purpose, we isolated AA BM-MSCs through whole bone marrow cell culture, evaluated a series of small molecule drugs using the in vitro adipogenic differentiation model of BM-MSCs, and finally focused on emodin, a natural anthraquinone derivative. Subsequently, we systematically investigated the molecular mechanism of emodin in attenuating adipogenic process by means of microarray profiling, bioinformatics analysis and lentivirus-mediated functional studies and rescue assay. RESULTS: We found that emodin presented significantly suppressive effect on the in vitro adipogenic differentiation of AA BM-MSCs. Further mechanistic investigation revealed that emodin could increase the expression of Tribbles homolog 3 (TRIB3) which exhibited remarkably decreased expression in AA BM-MSCs compared with the normal counterparts and was subsequently demonstrated as a negative regulator in adipogenesis of AA BM-MSCs. Besides, TRIB3 depletion alleviated the suppressive effect of emodin on the adipogenic differentiation of AA BM-MSCs. CONCLUSION: Our findings propose that emodin mediated TRIB3 up-regulation alleviates the adipogenic capacity of AA BM-MSCs, and emodin could serve as a potential therapeutic regimen for AA therapy.
9Melatonin Treatment Improves Mesenchymal Stem Cells Therapy by Preserving Stemness during Long-term In Vitro Expansion.PubMed
Yi Shuai, Li Liao, Xiaoxia Su, et al.
Mesenchymal stem cells (MSCs) are promising candidates for tissue regeneration and disease treatment. However, long-term in vitro passaging leads to stemness loss of MSCs, resulting in failure of MSCs therapy. Here, we report a melatonin-based strategy to improve cell therapy of in vitro cultured MSCs. Among four small molecules with anti-aging and stem cell-protection properties (rapamycin, resveratrol, quercetin and melatonin), colony forming, proliferation, and osteogenic differentiation assay showed that melatonin was the most efficient to preserve self-renewal and differentiation properties of rat bone marrow MSCs (BMMSCs) after long-term passaging. Functional assays confirmed melatonin treatment did not affect the colony forming, proliferation and osteogenic differentiation of BMMSCs cultured for 1 or 4 passages, but largely prevented the decline of self-renew and differentiation capacity of BMMSCs cultured for 15 passages in vitro. Furthermore, heterotopic osteogenesis assay, critical size calvarial defects repair assay, osteoporosis treatment and experimental colitis therapy assay strongly certified that melatonin preserved the therapeutic effect of long-term passaged BMMSCs on bone regeneration and immunotherapy in vivo. Mechanistically, melatonin functioned by activating antioxidant defense system, inhibiting the pathway of cell senescence, and preserving the expression of gene governing the stemness. Taken together, our findings showed that melatonin treatment efficiently prevented the dysfunction and therapeutic failure of BMMSCs after long-term passaging, providing a practical strategy to improve the application of BMMSCs in tissue engineering and cytotherapy.
10Multi-lineage MSC differentiation via engineered morphogen fields.PubMed
P R Arany, G X Huang, O Gadish, et al.
Tissue loss due to oral diseases requires the healing and regeneration of tissues of multiple lineages. While stem cells are native to oral tissues, a current major limitation to regeneration is the ability to direct their lineage-specific differentiation. This work utilizes polymeric scaffold systems with spatiotemporally controlled morphogen cues to develop precise morphogen fields to direct mesenchymal stem cell differentiation. First, a simple three-layer scaffold design was developed that presented two spatially segregated, lineage-specific cues (Dentinogenic TGF-β1 and Osteogenic BMP4). However, this system resulted in diffuse morphogen fields, as assessed by the in vitro imaging of cell-signaling pathways triggered by the morphogens. Mathematical modeling was then exploited, in combination with incorporation of specific inhibitors (neutralizing antibodies or a small molecule kinase inhibitor) into each morphogen in an opposing spatial pattern as the respective morphogen, to design a five-layer scaffold that was predicted to yield distinct, spatially segregated zones of morphogen signaling. To validate this system, undifferentiated MSCs were uniformly seeded in these scaffold systems, and distinct mineralized tissue differentiation were noted within these morphogen zones. Finally, to demonstrate temporal control over morphogen signaling, latent TGF-β1 was incorporated into one region of a concentric scaffold design, and laser treatment was used to activate the morphogen on-demand and to induce dentin differentiation solely within that specific spatial zone. This study demonstrates a significant advance in scaffold design to generate precise morphogen fields that can be used to develop in situ models to explore tissue differentiation and may ultimately be useful in engineering multi-lineage tissues in clinical dentistry.
11Tunable hydrogels for mesenchymal stem cell delivery: Integrin-induced transcriptome alterations and hydrogel optimization for human wound healing.PubMed
Alina I Marusina, Alexander A Merleev, Jesus I Luna, et al.
Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.
12Nanofunctionalized Microparticles for Glucose Delivery in Three-Dimensional Cell Assemblies.PubMed
Maria G Fois, Aygul Zengin, Ke Song, et al.
Three-dimensional (3D) cell assemblies, such as multicellular spheroids, can be powerful biological tools to closely mimic the complexity of cell-cell and cell-matrix interactions in a native-like microenvironment. However, potential applications of large spheroids are limited by the insufficient diffusion of oxygen and nutrients through the spheroids and, thus, result in the formation of a necrotic core. To overcome this drawback, we present a new strategy based on nanoparticle-coated microparticles. In this study, microparticles function as synthetic centers to regulate the diffusion of small molecules, such as oxygen and nutrients, within human mesenchymal stem cell (hMSC) spheroids. The nanoparticle coating on the microparticle surface acts as a nutrient reservoir to release glucose locally within the spheroids. We first coated the surface of the poly(lactic--glycolic acid) (PLGA) microparticles with mesoporous silica nanoparticles (MSNs) based on electrostatic interactions and then formed cell-nanofunctionalized microparticle spheroids. Next, we investigated the stability of the MSN coating on the microparticles' surface during 14 days of incubation in cell culture medium at 37 °C. Then, we evaluated the influence of MSN-coated PLGA microparticles on spheroid aggregation and cell viability. Our results showed the formation of homogeneous spheroids with good cell viability. As a proof of concept, fluorescently labeled glucose (2-NBD glucose) was loaded into the MSNs at different concentrations, and the release behavior was monitored. For cell culture studies, glucose was loaded into the MSNs coated onto the PLGA microparticles to sustain local nutrient release within the hMSC spheroids. results demonstrated that the local delivery of glucose from MSNs enhanced the cell viability in spheroids during a short-term hypoxic culture. Taken together, the newly developed nanofunctionalized microparticle-based delivery system may offer a versatile platform for local delivery of small molecules within 3D cellular assemblies and, thus, improve cell viability in spheroids.
13The Different Molecular Code in Generation of Dopaminergic Neurons from Astrocytes and Mesenchymal Stem Cells.PubMed
Nana Wang, Xingrui Ji, Yue Wu, et al.
Transplantation of exogenous dopaminergic (DA) neurons is an alternative strategy to replenish DA neurons that have lost along the course of Parkinson's disease (PD). From the perspective of ethical acceptation, the source limitations, and the intrinsic features of PD pathology, astrocytes (AS) and mesenchymal stem cells (MSCs) are the two promising candidates of DA induction. In the present study, we induced AS or MSCs primary culture by the combination of the classical transcription-factor cocktails Mash1, Lmx1a, and Nurr1 (MLN), the chemical cocktails (S/C/D), and the morphogens SHH, FGF8, and FGF2 (S/F8/F2); the efficiency of induction into DA neurons was further analyzed by using immunostaining against the DA neuronal markers. AS could be efficiently converted into the DA neurons in vitro by the transcriptional regulation of MLN, and the combination with S/C/D or S/F8/F2 further increased the conversion efficiency. In contrast, MSCs from umbilical cord (UC-MSCs) or adipose tissue (AD-MSCs) showed moderate TH immunoreactivity after the induction with S/F8/F2 instead of with MLN or S/C/D. Our data demonstrated that AS and MSCs held lineage-specific molecular codes on the induction into DA neurons and highlighted the unique superiority of AS in the potential of cell replacement therapy for PD.
14Optimisation of a Method for the Differentiation of Human Umbilical Cord-derived Mesenchymal Stem Cells Toward Renal Epithelial-like Cells.PubMed
Rakhshinda Habib, Shumaila Fahim, Mohsin Wahid, et al.
Human umbilical cord-derived mesenchymal stem cells (hucMSCs) can differentiate into multiple cell lineages, but few methods have been developed to generate kidney lineage cells. Due to their human origin, pluripotent nature and immunomodulatory properties, these stem cells are attractive candidates for clinical applications such as the repair or regeneration of damaged organs. This study evaluated the renal differentiation potential of hucMSCs, when exposed for 10 days to optimised concentrations of retinoic acid, activin-A and bone morphogenetic protein-7 (BMP-7) in various combinations, with and without the priming of the cells with a Wnt signalling pathway activator (CHIR99021). The hucMSCs were isolated and characterised according to surface marker expression (CD73, CD90, CD44, CD146 and CD8) and tri-lineage differentiation potential. The expression of key marker genes (, , , , , and ) was examined by qRT-PCR. Specific marker protein expression (E-cadherin, cytokeratin-8 and cytokeratin-19) was analysed by immunocytochemistry. CHIR99021-primed cells treated with the retinoic acid, activin-A and BMP-7 cocktail showed epithelial cell-like differentiation - i.e. distinct phenotypic changes, as well as upregulated gene and protein expression, were observed that were consistent with an epithelial cell phenotype. Thus, our results showed that hucMSCs can efficiently differentiate into renal epithelial-like cells. This work may help in the development of focused therapeutic strategies, in which lineage-defined human stem cells can be used for renal regeneration.
15A Modular Approach to Sensitized Two-Photon Patterning of Photodegradable Hydrogels.PubMed
Markus Lunzer, Liyang Shi, Orestis G Andriotis, et al.
Photodegradable hydrogels have emerged as useful platforms for research on cell function, tissue engineering, and cell delivery as their physical and chemical properties can be dynamically controlled by the use of light. The photo-induced degradation of such hydrogel systems is commonly based on the integration of photolabile o-nitrobenzyl derivatives to the hydrogel backbone, because such linkers can be cleaved by means of one- and two-photon absorption. Herein we describe a cytocompatible click-based hydrogel containing o-nitrobenzyl ester linkages between a hyaluronic acid backbone, which is photodegradable in the presence of cells. It is demonstrated for the first time that by using a cyclic benzylidene ketone-based small molecule as photosensitizer the efficiency of the two-photon degradation process can be improved significantly. Biocompatibility of both the improved two-photon micropatterning process as well as the hydrogel itself is confirmed by cell culture studies.
163D printing of recombinant collagen/chitosan methacrylate/nanoclay hydrogels loaded with Kartogenin nanoparticles for cartilage regeneration.PubMed
Wanting Zhang, Kejia Shi, Jianfeng Yang, et al.
Cartilage defects are frequently caused by trauma, illness and degradation of the cartilage. If these defects are not sufficiently treated, the joints will degrade irreversibly, possibly resulting in disability. Articular cartilage lacks blood vessels and nerves and is unable to regenerate itself, so the repair of cartilage defects is extremely challenging in clinical treatment. Tissue engineering technology is an emerging technology in cartilage repair and cartilage regeneration. 3D-printed hydrogels show great potential in cartilage tissue engineering for the fabrication of 3D cell culture scaffolds to mimic extracellular matrix. In this study, we construct a 3D-printed hydrogel loaded with nanoparticles by electrostatic interaction and photo cross-linking for the regeneration of cartilage, which has adaptable and drug-continuous release behavior. A photopolymerizable bioink was prepared using recombinant collagen, chitosan, nanoclay Laponite-XLG and nanoparticles loaded with Kartogenin (KGN). This bioink was added with KGN, a small molecule drug that promotes cartilage differentiation, and as a result, the 3D-printed CF/CM/3%LAP/KGN scaffolds obtained by extrusion printing is expected to be used for cartilage repair. It was shown that the 3D-printed scaffolds had good cytocompatibility for human bone marrow mesenchymal stem cells (hBMSCs) and exhibited excellent antimicrobial properties, the continuous release of KGN in the scaffold induced the hBMSCs differentiation into chondrocytes, which significantly enhanced the expression of collagen II and glycosaminoglycan. studies have shown that implantation of KGN-loaded scaffolds into cartilage-injured tissues promoted cartilage tissue regeneration. This study demonstrated that 3D-printed CF/CM/3%LAP/KGN scaffolds can be used for cartilage repair, which is expected to lead to new healing opportunities for cartilage injury-based diseases.
17Stem cell library screen identified ruxolitinib as regulator of osteoblastic differentiation of human skeletal stem cells.PubMed
Nihal AlMuraikhi, Dalia Ali, Aliah Alshanwani, et al.
BACKGROUND: Better understanding of the signaling pathways that regulate human bone marrow stromal stem cell (hBMSC) differentiation into bone-forming osteoblasts is crucial for their clinical use in regenerative medicine. Chemical biology approaches using small molecules targeting specific signaling pathways are increasingly employed to manipulate stem cell differentiation fate. METHODS: We employed alkaline phosphatase activity and staining assays to assess osteoblast differentiation and Alizarin R staining to assess mineralized matrix formation of cultured hBMSCs. Changes in gene expression were assessed using an Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. For in vivo ectopic bone formation experiments, hMSCs were mixed with hydroxyapatite-tricalcium phosphate granules and implanted subcutaneously into the dorsal surface of 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius Red staining were used to detect bone formation in vivo. RESULTS: We identified several compounds which inhibited osteoblastic differentiation of hMSCs. In particular, we identified ruxolitinib (INCB018424) (3 μM), an inhibitor of JAK-STAT signaling that inhibited osteoblastic differentiation and matrix mineralization of hMSCs in vitro and reduced ectopic bone formation in vivo. Global gene expression profiling of ruxolitinib-treated cells identified 847 upregulated and 822 downregulated mRNA transcripts, compared to vehicle-treated control cells. Bioinformatic analysis revealed differential regulation of multiple genetic pathways, including TGFβ and insulin signaling, endochondral ossification, and focal adhesion. CONCLUSIONS: We identified ruxolitinib as an important regulator of osteoblast differentiation of hMSCs. It is plausible that inhibition of osteoblast differentiation by ruxolitinib may represent a novel therapeutic strategy for the treatment of pathological conditions caused by accelerated osteoblast differentiation and mineralization.
18Small molecule mesengenic induction of human induced pluripotent stem cells to generate mesenchymal stem/stromal cells.PubMed
Yen Shun Chen, Rebecca A Pelekanos, Rebecca L Ellis, et al.
The translational potential of mesenchymal stem/stromal cells (MSCs) is limited by their rarity in somatic organs, heterogeneity, and need for harvest by invasive procedures. Induced pluripotent stem cells (iPSCs) could be an advantageous source of MSCs, but attempts to derive MSCs from pluripotent cells have required cumbersome or untranslatable techniques, such as coculture, physical manipulation, sorting, or viral transduction. We devised a single-step method to direct mesengenic differentiation of human embryonic stem cells (ESCs) and iPSCs using a small molecule inhibitor. First, epithelial-like monolayer cells were generated by culturing ESCs/iPSCs in serum-free medium containing the transforming growth factor-β pathway inhibitor SB431542. After 10 days, iPSCs showed upregulation of mesodermal genes (MSX2, NCAM, HOXA2) and downregulation of pluripotency genes (OCT4, LEFTY1/2). Differentiation was then completed by transferring cells into conventional MSC medium. The resultant development of MSC-like morphology was associated with increased expression of genes, reflecting epithelial-to-mesenchymal transition. Both ESC- and iPSC-derived MSCs exhibited a typical MSC immunophenotype, expressed high levels of vimentin and N-cadherin, and lacked expression of pluripotency markers at the protein level. Robust osteogenic and chondrogenic differentiation was induced in vitro in ES-MSCs and iPS-MSCs, whereas adipogenic differentiation was limited, as reported for primitive fetal MSCs and ES-MSCs derived by other methods. We conclude that treatment with SB431542 in two-dimensional cultures followed by culture-induced epithelial-to-mesenchymal transition leads to rapid and uniform MSC conversion of human pluripotent cells without the need for embryoid body formation or feeder cell coculture, providing a robust, clinically applicable, and efficient system for generating MSCs from human iPSCs.
19Exosomes Derived from Human Bone Marrow Mesenchymal Stem Cells Promote Tumor Growth Through Hedgehog Signaling Pathway.PubMed
Jin Qi, Yali Zhou, Zuoyi Jiao, et al.
BACKGROUND/AIMS: Mesenchymal stem/stromal cells (MSCs) are known to home to sites of tumor microenvironments where they participate in the formation of the tumor microenvironment and to interplay with tumor cells. However, the potential functional effects of MSCs on tumor cell growth are controversial. Here, we, from the view of bone marrow MSC-derived exosomes, study the molecular mechanism of MSCs on the growth of human osteosarcoma and human gastric cancer cells. METHODS: MSCs derived from human bone marrow (hBMSCs) were isolated and cultured in complete DMEM/F12 supplemented with 10% exosome-depleted fetal bovine serum and 1% penicillin-streptomycin, cell culture supernatants containing exosomes were harvested and exosome purification was performed by ultracentrifugation. Osteosarcoma (MG63) and gastric cancer (SGC7901) cells, respectively, were treated with hBMSC-derived exosomes in the presence or absence of a small molecule inhibitor of Hedgehog pathway. Cell viability was measured by transwell invasion assay, scratch migration assay and CCK-8 test. The expression of the signaling molecules Smoothened, Patched-1, Gli1 and the ligand Shh were tested by western blot and RT-PCR. RESULTS: In this study, we found that hBMSC-derived exosomes promoted MG63 and SGC7901 cell growth through the activation of Hedgehog signaling pathway. Inhibition of Hedgehog signaling pathway significantly suppressed the process of hBMSC-derived exosomes on tumor growth. CONCLUSION: Our findings demonstrated the new roles of hedgehog signaling pathway in the hBMSCs-derived exosomes induced tumor progression.
20Exposure to small molecule cocktails allows induction of neural crest lineage cells from human adipose-derived mesenchymal stem cells.PubMed
Yuzo Takayama, Yuka Akagi, Yoichiro Shibuya, et al.
Neural crest cells (NCCs) are a promising source for cell therapy and regenerative medicine owing to their multipotency, self-renewability, and capability to secrete various trophic factors. However, isolating NCCs from adult organs is challenging, because NCCs are broadly distributed throughout the body. Hence, we attempted to directly induce NCCs from human adipose-derived mesenchymal stem cells (ADSCs), which can be isolated easily, using small molecule cocktails. We established a controlled induction protocol with two-step application of small molecule cocktails for 6 days. The induction efficiency was evaluated based on mRNA and protein expression of neural crest markers, such as nerve growth factor receptor (NGFR) and sex-determining region Y-box 10 (SOX10). We also found that various trophic factors were significantly upregulated following treatment with the small molecule cocktails. Therefore, we performed global profiling of cell surface makers and identified distinctly upregulated markers, including the neural crest-specific cell surface markers CD271 and CD57. These results indicate that our chemical treatment can direct human ADSCs to developing into the neural crest lineage. This offers a promising experimental platform to study human NCCs for applications in cell therapy and regenerative medicine.