Hydrogel Microwell Arrays Research Articles

Artificial niche microarrays for probing single stem cell fate in high throughput

To understand the regulatory role of niches in maintaining stem-cell fate, multifactorial in vitro models are required. These systems should enable analysis of biochemical and biophysical niche effectors in a combinatorial fashion and in the context of a physiologically relevant cell-culture substrate. We report a microengineered platform comprised of soft hydrogel microwell arrays with modular stiffness (shear moduli of 1-50 kPa) in which individual microwells can be functionalized with combinations of proteins spotted by robotic technology. To validate the platform, we tested the effect of cell-cell interactions on adipogenic differentiation of adherent human mesenchymal stem cells (MSCs) and the effect of substrate stiffness on osteogenic MSC differentiation. We also identified artificial niches supporting extensive self-renewal of nonadherent mouse neural stem cells (NSCs). Using this method, it is possible to probe the effect of key microenvironmental perturbations on the fate of any stem cell type in single cells and in high throughput.

Preparation and Characterization of Genetically Engineered Mesenchymal Stem Cell Aggregates for Regenerative Medicine

Combining cell- and gene-based therapy is a promising therapeutic strategy in regenerative medicine. The aim of this study was to develop genetically modified mesenchymal stem cell (MSC) aggregates using a poly(ethylene gly- col) (PEG) hydrogel micro-well array technique. Stable PEG hydrogel micro-well arrays with diameters of 200 to 500 µm were fabricated and used to generate genetically engineered MSC aggregates. Rat bone marrow-derived MSCs were trans- fected with a green fluorescent protein (GFP) plasmid as a reporter gene, and aggregated by culturing in the PEG hydrogel micro-well arrays. The resultant cell aggregates had a mean diameter of less than 200 µm, and maintained the mesenchymal phenotype even after genetic modification and cell aggregation. Transplantation of MSC aggregates that are genetically modified to express therapeutic or cell-survival genes may be a potential therapeutic approach for regenerative medicine. Key wordsCell and gene therapy, Mesenchymal stem cells, Micro-well array, Cell aggregates

Single Cell Phospho-Flow Analysis of Cytokine Stimulation in Human Hematopoietic Progenitors Reveals That G-CSF Acts Directly On Human Hematopoietic Stem Cells.

Abstract 3617Poster Board III-553Hematopoietic stem and progenitor cells (HSPC) give rise to all blood cell lineages for the lifetime of an individual. However, their low frequency has precluded the use of traditional biochemical methods to study their signaling biology. We developed a phospho-specific flow cytometry approach that allows for the simultaneous interrogation of multiple elements of signaling networks in primary human HSPC at the single cell level without prospective purification by cell sorting. We found that HSPC contain biochemically distinct signaling subpopulations, and that, although considered quiescent, HSC from cord blood and adult bone marrow respond directly to several cytokine stimuli through multiple signaling nodes including JAK/Stat, Erk, and Akt. Surprisingly, we found that a subset of HSC respond rapidly and directly to in vitro G-CSF stimulation through Stat3 signaling. The addition of G-CSF to purified cultures of HSC significantly increased the percentage of cells that had entered the cell cycle. Moreover, when G-CSF was added to cultures of purified human HSC spatially segregated in hydrogel microwell arrays, we observed a significant increase in the frequency of HSC proliferation. These results demonstrate that contrary to the accepted paradigm, G-CSF acts directly on human HSC. Cytometric single cell characterization of the molecular behavior of HSPC in a wide variety of hematopoietic malignancies can now be applied to study the signaling behavior of other primary and rare stem cell populations. Disclosures:Weissman:U.S. Patent Application 11/528,890 entitled “Methods for Diagnosing and Evaluating Treatment of Blood Disorders.”: Patents & Royalties; Stem Cells Inc.: Cofounder and Director; Cellerant Inc.: Cofounder; Amgen: Equity Ownership. Nolan:BD Biosciences: Consultancy.

A microfluidic chip with poly(ethylene glycol) hydrogel microarray on nanoporous alumina membrane for cell patterning and drug testing

In this paper, a microfluidic silicon chip with poly(ethylene glycol) (PEG) hydrogel microarray on the nanoporous anodized aluminum oxide (AAO) membrane was fabricated to form cell based microarray with controlled drug delivery. The PEG hydrogel microstructures were fabricated using photolithography on nanoporous alumina surface modified with a 3-(trimethoxysilyl)propyl methacrylate (TPM) monolayer. During the photopolymerization reaction, 10 × 10 hydrogel microwell arrays were covalently bonded to the substrate via the TPM monolayer for cell patterning. Human KYSE-30 esophageal squamous epithelial cancer cells were shown to selectively adhere on the TPM modified alumina surface inside the microwells. The diffusion studies for anti-cancer drug molecule of cisplatin were carried out for the microfabricated membrane array using a mini-diffusion chamber. The diffusion curves for pore size of 25 nm and 55 nm showed different release profiles. Then, the drug testing experiment with cisplatin was explored using the microfabricated membrane array with pore size of 55 nm for testing the drug effects on the KYSE-30 cancer cells. The cytotoxic effects of cisplatin on cancer cell number and cell morphology within microwells were explored based on this cell microarray.

Microwell-mediated control of embryoid body size regulates embryonic stem cell fate via differential expression of WNT5a and WNT11

Recently, various approaches for controlling the embryonic stem (ES) cell microenvironment have been developed for regulating cellular fate decisions. It has been reported that the lineage specific differentiation could be affected by the size of ES cell colonies and embryoid bodies (EBs). However, much of the underlying biology has not been well elucidated. In this study, we used microengineered hydrogel microwells to direct ES cell differentiation and determined the role of WNT signaling pathway in directing the differentiation. This was accomplished by forming ES cell aggregates within microwells to form different size EBs. We determined that cardiogenesis was enhanced in larger EBs (450 microm in diameter), and in contrast, endothelial cell differentiation was increased in smaller EBs (150 microm in diameter). Furthermore, we demonstrated that the EB-size mediated differentiation was driven by differential expression of WNTs, particularly noncanonical WNT pathway, according to EB size. The higher expression of WNT5a in smaller EBs enhanced endothelial cell differentiation. In contrast, the increased expression of WNT11 enhanced cardiogenesis. This was further validated by WNT5a-siRNA transfection assay and the addition of recombinant WNT5a. Our data suggest that EB size could be an important parameter in ES cell fate specification via differential gene expression of members of the noncanonical WNT pathway. Given the size-dependent response of EBs to differentiate to endothelial and cardiac lineages, hydrogel microwell arrays could be useful for directing stem cell fates and studying ES cell differentiation in a controlled manner.

Perturbation of single hematopoietic stem cell fates in artificial niches

Hematopoietic stem cells (HSCs) are capable of extensive self-renewal in vivo and are successfully employed clinically to treat hematopoietic malignancies, yet are in limited supply as in culture this self-renewal capacity is lost. Using an approach at the interface of stem cell biology and bioengineering, here we describe a novel platform of hydrogel microwell arrays for assessing the effects of either secreted or tethered proteins characteristic of the in vivo microenvironment, or niche, on HSC fate in vitro. Time-lapse microscopic analyses of single cells were crucial to overcoming inevitable heterogeneity of FACS-enriched HSCs. A reduction in proliferation kinetics or an increase in asynchronous division of single HSCs in microwells in response to specific proteins (Wnt3a and N-Cadherin) correlated well with subsequent serial long-term blood reconstitution in mice in vivo. Single cells that divided once in the presence of a given protein were capable of in vivo reconstitution, providing evidence of self-renewal divisions of HSCs in vitro. These results validate the hydrogel microwell platform as a broadly applicable paradigm for dissecting the regulatory role of specific signals within a complex stem cell niche.

Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays

The neurosphere assay is the standard retrospective assay to test the self-renewal capability and multipotency of neural stem cells (NSCs) in vitro. However, it has recently become clear that not all neurospheres are derived from a NSC and that on conventional cell culture substrates, neurosphere motility may cause frequent neurosphere "merging" [Nat Methods 2006;3:801-806; Stem Cells 2007;25:871-874]. Combining biomimetic hydrogel matrix technology with microengineering, we developed a microwell array platform on which NSC fate and neurosphere formation can be unequivocally attributed to a single founding cell. Using time-lapse microscopy and retrospective immunostaining, the fate of several hundred single NSCs was quantified. Compared with conventional neurosphere culture methods on plastic dishes, we detected a more than 100% increase in single NSC viability on soft hydrogels. Effective confinement of single proliferating cells to microwells led to neurosphere formation of vastly different sizes, a high percentage of which showed stem cell phenotypes after one week in culture. The reliability and increased throughput of this platform should help to better elucidate the function of sphere-forming stem/progenitor cells independent of their proliferation dynamics. Disclosure of potential conflicts of interest is found at the end of this article.