KRT5high TP63-expressing urothelial basal cells act as a driver to bladder urothelium regeneration in rabbit

Abstract

Background  Urothelial regeneration is a crucial part of bladder tissue engineering. However, there is a lack of ideal “seed cells” in current practices. Here, we demonstrated that a sub-population of p63 positive basal cells could be activated and differentiate into intermediate and superficial umbrella cells after full-thickness mucosal resection in rabbit. Methods  A focal mucosal resection model was used to characterize the role of different urothelial cells during regeneration. Urothelial basal cells were isolated from rabbit bladder mucosa and cultured in vitro. The basal cells were then transplanted in vivo in a manner of cell sheet for reconstruction. Results  Via single-cell RNA sequencing (scRNA-seq), it has been confirmed that the cluster of KRT5high TP63-expressing cells possesses a ‘stemness’ signature which can give rise to lineage cell types sequentially. With a strong support from the underneath pre-set capsule vascular bed, the transplanted cell sheet could develop into a physio-morphology resembled to the native mucosa in vivo. Importantly, we validated that the bioengineered urothelium implemented perfect barrier function after implanted to bladder. Conclusions  In summary, bioengineering urothelium with KRT5high TP63-expressing basal cells on a capsule vascular bed offers a promising strategy for bladder tissue engineering and provides a model for drug screening and bladder disease research.

1. Introduction

The bladder lumen is lined by urothelium, a transitional epithelium consisting of three cell types: superficial umbrella cell, intermediate cell and basal cell [1]. The primary role of urothelium is to generate a robust barrier to protect underlying tissue from toxic substances within hypertonic urine. Bladder reconstruction is desired in various clinical scenarios, such as bladder cancer, congenital malformations, neuropathic bladder, trauma, infection or inflammation [2]. Currently, gastrointestinal segments remain the primary source of material for bladder reconstruction, however, the transposed intestine segments maintain an absorptive and mucus-producing epithelium after replacing the bladder, which lead to numerous clinical complications including metabolic disturbance, stone formation, chronic infections and secondary malignancies [3].

As the field of tissue engineering advances, bioengineered bladder is expected to replace gastrointestinal tissue as a new gold standard for bladder reconstruction. Although the generation of a bioengineered tissue with a morphology similar to that of the native bladder has already been accomplished, the restoration of a functional bladder using this strategy has not been achieved so far, which is hindering the application of bioengineered bladder in clinical settings. Urothelial regeneration is a crucial part of bladder tissue engineering as any leakage of urine into the underlying tissues may provoke a fibrotic outcome associated with randomly organized collagen fibres, muscle contraction, and compromised bladder capacity [4]. Thus, to establish a strategy enabling efficient bladder full-thickness reconstruction with functional urothelium is of clinical importance, particularly in cases requiring to construct a neobladder.