Introduction: Identification and purification of mesenchymal stem cells (MSCs) expanded in culture for therapeutic use is crucial for improved yield and optimal results. Fibroblasts are the most common cell type in connective tissue and are commonly found as contaminants of MSC cultures, affecting cell yield and potentially causing tumour formation after cell transplantation. In the current study, we wished to identify cell surface markers that can differentiate MSCs of different origins from fibroblasts.Material and methods: Mesenchymal stem cells were isolated from bone marrow, adipose tissue, Wharton’s jelly, and placental tissue, and fibroblasts were isolated from foreskin (as a negative control) in order to examine the differences in the expression of a panel of 14 different cell surface markers using multiplex flow cytometry.Results: Our results indicate that the following markers could be useful in differentiating between fibroblasts and MSCs derived from the following: adipose tissue – CD79a, CD105, CD106, CD146, and CD271; Wharton’s jelly – CD14, CD56, and CD105; bone marrow – CD105, CD106, and CD146; and placental tissue – CD14, CD105, and CD146. Furthermore, we found that, contradictory to previous studies, CD26 is not fibroblast specific.Conclusions: The results of our study indicate that cell surface markers may prove to be a useful tool in the discrimination between MSCs of different origins and fibroblasts, and thus may be used to authenticate the identity of the isolated cells.

The stemness in keratinocyte stem cells (KSCs) is determined by their gene expression patterns. KSCs are crucial in maintaining epidermal homeostasis and wound repair and are widely used candidates for therapeutic applications. Although several studies have reported their positive identifiers, unique biomarkers for KSCs remain elusive. Here, we aim to identify potential candidate stem cell markers. Human epidermal keratinocytes (HEKs) from neonatal foreskin tissues were isolated and cultured. Single-cell clonal analysis identified and characterized three types of cells: KSCs (holoclones), transient amplifying cells (TACs; meroclones), and differentiated cells (DSCs; paraclones). The clonogenic potential of KSCs demonstrated the highest proliferation potential of KSCs, followed by TACs and DSCs, respectively. Whole-transcriptome analysis using microarray technology unraveled the molecular signatures of these cells. These results were validated by quantitative real-time polymerase chain reaction and flow cytometry analysis. A total of 301 signature upregulated and 149 downregulated differentially expressed genes (DEGs) were identified in the KSCs, compared to TACs and DSCs. Furthermore, DEG analyses revealed new sets of genes related to cell proliferation, cell adhesion, surface makers, and regulatory factors. In conclusion, this study provides a useful source of information for the identification of potential SC-specific candidate markers.

As the field of three-dimensional (3D) bioprinting gains increased momentum, complex 3D bioprinters are being developed to keep up with the needs of biofabrication and tissue engineering. Cartesian-based linear 3D bioprinters have facilitated the fabrication of 3D biological constructs and scaffolds. However, to achieve meaningful advancement in biofabrication, 3D bioprinters need increased freedom of motion, seamless multi-material printing, full automation, and ease of use. In this paper, we propose TwinPrint, a dual-arm robotic 3D bioprinting system, designed to be compatible with soft bioinks to build multi-material constructs, crucial for creating functional tissue. The uniquely integrated robotic 3D bioprinter—comprising an in-house fabricated coaxial nozzle, two 4-axis robotic arms, six microfluidic pumps, and a software interface—work harmoniously as a single unit. We showcase the development of the Python-based software and Graphical User Interface, integrating all components into a single easy-to-use interface. Due to their human-like and instantaneous gelation properties, peptide-based bioinks were used as printing material to test the system. Developed in our laboratory as an alternative to gelatin- and alginate-based bioinks, they avoided chemical and ultraviolet-crosslinking by solidifying instantaneously under physiological conditions. For system performance testing, acellular and cellular constructs were observed for structural fidelity, multi-material layering, printing accuracy, cell viability, and proliferation. Overall, our proposed system showcases an innovative integration of robotics for biofabrication to expedite the printing process and enable multi-task print protocols. By saving time, increasing degrees of freedom, and expanding printing complexity, we believe TwinPrint is a promising next step for biofabrication.

This book chapter provides an in-depth overview of the clinical relevance of mesenchymal stem cells (MSCs) derived from various sources, highlighting the importance of whole transcriptome analysis in revealing their inherent potential. The chapter delves into different sources of MSCs, such as bone marrow, adipose tissue, umbilical cord, and placenta, and compares their respective properties and capabilities. Additionally, it explores the latest advancements in whole transcriptome analysis, including RNA sequencing and microarray analysis, and their applications in MSC research. The aim is to provide a comprehensive understanding of how high-end technologies, such as whole transcriptome analysis, can aid in identifying the inherent potential of cells for therapeutic applications. It will also discuss how such gene expression approaches helped identify the inherent potential of specific MSC sources, tailoring their use towards specific clinical applications, including immune tolerance and modulation, osteogenesis, and chondrogenesis. Additionally, it highlights the importance of extracellular vesicles derived from MSCs. This knowledge will be beneficial for researchers and clinicians working towards developing MSC-based treatments for regenerative medicine and cellular therapy.