Optimization of seeding cell density for differentiation of adipose-derived stem cells into epithelial-like cells on bioengineered composite scaffolds

IF 2.2 3区生物学 Q4 CELL BIOLOGY

Differentiation Pub Date : 2025-05-01 DOI:10.1016/j.diff.2025.100870

Yourka D. Tchoukalova , Manisha K. Shah , Cheryl E. Myers , Nan Zhang , David G. Lott

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引用次数: 0

Abstract

This study investigates the biological factors influencing the epithelial differentiation of adipose-derived stem cells (ASC) to develop an engineered upper airway construct. One fraction of ASC was seeded onto a fibrin sealant (Tisseel) matrix encompassing an additional equal fraction of ASC that has been integrated into a porous polyethylene scaffold (Medpor®). Constructs with ASC seeded at total densities of 5 × 10⁵, 1 × 10⁶, 2.5 × 10⁶, and 5 × 10⁶ cells cm-2 were cultured under submerged conditions for 11 days to achieve partial epithelial differentiation (PD). To simulate post-transplantation exposure to air and interaction with host epithelial cells, PD constructs with ASC at 5 × 10⁶ cells cm-2 were transitioned to air-liquid interface (ALI) conditions for additional 10 days (PD/ALI-21d) or 21 days (PD/ALI-32d). The latter cultures were either maintained alone or co-cultured with bronchial epithelial cells (PD/ALI-32d + BEAS). Gene expressions of mesenchymal and epithelial basal, secretory, and ciliated cell markers were assessed and validated via immunohistochemistry.

ASC seeded at 5 × 10⁶ cells cm-2 achieved the highest partial epithelial differentiation, supporting the use of this density for further experiments. In PD/ALI-21d, basal and secretory epithelial marker gene expression significantly increased, while ciliated cell markers remained unchanged. In PD/ALI-32d, expression of basal and goblet cell markers and several mesenchymal stem cell markers decreased, but co-culturing with BEAS maintained the levels of their expression. These results indicate that long-term ALI cultures cannot sustain terminal differentiation of ASC into secretory phenotypes without co-culture with primary epithelial cells.

In conclusion, partially differentiated ASC on constructs maintain a stem cell phenotype and may promote differentiation into basal/secretory phenotypes, but not ciliated cells. Enhancing ciliogenesis and ensuring ASC commitment to the epithelial lineage, require modifications to the study design.

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生物工程复合支架上脂肪干细胞向上皮样细胞分化的种子细胞密度优化

本研究探讨了影响脂肪源性干细胞（ASC）上皮细胞分化的生物学因素，以形成工程化的上呼吸道结构。将一部分ASC植入纤维蛋白密封胶（Tisseel）基质中，其中包含另外等量的ASC， ASC已集成到多孔聚乙烯支架（Medpor®）中。总密度分别为5 × 105、1 × 106、2.5 × 106和5 × 106细胞cm-2的ASC构建体在潜水条件下培养11天，实现部分上皮分化（PD）。为了模拟移植后暴露于空气中并与宿主上皮细胞相互作用，将5 × 106细胞cm-2的ASC构建物转移到气液界面（ALI）条件下再增加10天（PD/ALI-21d）或21天（PD/ALI-32d）。后一种培养物单独维持或与支气管上皮细胞（PD/ALI-32d + BEAS）共培养。通过免疫组织化学评估和验证间充质和上皮基底细胞、分泌细胞和纤毛细胞标记物的基因表达。5 × 106个细胞cm-2的ASC种子获得了最高的部分上皮分化，支持使用该密度进行进一步的实验。在PD/ALI-21d中，基础和分泌上皮标记基因表达显著增加，而纤毛细胞标记基因表达不变。在PD/ALI-32d中，基底细胞和杯状细胞标记物以及几种间充质干细胞标记物的表达下降，但与BEAS共培养保持其表达水平。这些结果表明，如果不与原代上皮细胞共培养，长期ALI培养不能维持ASC向分泌型的最终分化。综上所述，部分分化的ASC维持了干细胞表型，并可能促进分化为基底/分泌型，而不是纤毛细胞。加强纤毛发生和确保ASC对上皮谱系的承诺，需要对研究设计进行修改。

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来源期刊

Differentiation 生物-发育生物学

CiteScore

4.10

自引率

3.40%

发文量

审稿时长

51 days

期刊介绍： Differentiation is a multidisciplinary journal dealing with topics relating to cell differentiation, development, cellular structure and function, and cancer. Differentiation of eukaryotes at the molecular level and the use of transgenic and targeted mutagenesis approaches to problems of differentiation are of particular interest to the journal. The journal will publish full-length articles containing original work in any of these areas. We will also publish reviews and commentaries on topics of current interest. The principal subject areas the journal covers are: • embryonic patterning and organogenesis • human development and congenital malformation • mechanisms of cell lineage commitment • tissue homeostasis and oncogenic transformation • establishment of cellular polarity • stem cell differentiation • cell reprogramming mechanisms • stability of the differentiated state • cell and tissue interactions in vivo and in vitro • signal transduction pathways in development and differentiation • carcinogenesis and cancer • mechanisms involved in cell growth and division especially relating to cancer • differentiation in regeneration and ageing • therapeutic applications of differentiation processes.