Comparative proteomic analysis of two distinct stem-cell populations from human amniotic fluid.

Human amniotic fluid (AF) contains a variety of stem cells of embryonic and extra-embryonic origins. We characterized two distinct types of stem cells isolated from residual AF material derived from prenatal diagnostic amniocentesis. The two types of cells differed in their morphology and growth kinetics, showing fast (fast human amniotic stem cells; fHASCs) or slow (slow human amniotic stem cells; sHASCs) population-doubling times. Both fHASCs and sHASCs expressed pluripotent stem-cell markers, yet unlike sHASCs, clonogenic fHASCs would generate embryoid bodies and maintain their original phenotype during prolonged in vitro passaging. fHASCs - but not sHASCs - expressed the KLF4, SSEA-4 and CD117 markers. Differential proteomic analysis allowed us to identify the protein patterns specific for either cell type as potentially contributing to their distinct phenotypes. We found thirty-six proteins that were differentially expressed by the two cell types, and those proteins were classified according to their biological and molecular functions. Bioinformatic cluster analysis revealed differential occurrence of cytoskeletal proteins, such as vimentin, F-actin-binding protein, and chloride intracellular channel protein 1. Selected proteins differentially expressed by fHASCs and sHASCs were further characterized by Western blot analysis and confocal microscopy.

Thermoresponsive worms for expansion and release of human embryonic stem cells.

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC.

Single-cell analysis of embryoid body heterogeneity using microfluidic trapping array.

The differentiation of pluripotent stem cells as embryoid bodies (EBs) remains a common method for inducing differentiation toward many lineages. However, differentiation via EBs typically yields a significant amount of heterogeneity in the cell population, as most cells differentiate simultaneously toward different lineages, while others remain undifferentiated. Moreover, physical parameters, such as the size of EBs, can modulate the heterogeneity of differentiated phenotypes due to the establishment of nutrient and oxygen gradients. One of the challenges in examining the cellular composition of EBs is the lack of analytical methods that are capable of determining the phenotype of all of the individual cells that comprise a single EB. Therefore, the objective of this work was to examine the ability of a microfluidic cell trapping array to analyze the heterogeneity of cells comprising EBs during the course of early differentiation. The heterogeneity of single cell phenotype on the basis of protein expression of the pluripotent transcription factor OCT-4 was examined for populations of EBs and single EBs of different sizes at distinct stages of differentiation. Results from the cell trap device were compared with flow cytometry and whole mount immunostaining. Additionally, single cells from dissociated pooled EBs or individual EBs were examined separately to discern potential differences in the value or variance of expression between the different methods of analysis. Overall, the analytical method described represents a novel approach for evaluating how heterogeneity is manifested in EB cultures and may be used in the future to assess the kinetics and patterns of differentiation in addition to the loss of pluripotency.

Extracellular matrix aggregates from differentiating embryoid bodies as a scaffold to support ESC proliferation and differentiation.

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications.