High-throughput analysis of signals regulating stem cell fate and function

Abstract

Stem cells exhibit promise in numerous areas of regenerative medicine. Their fate and function are governed by a combination of intrinsic determinants and signals from the local microenvironment, or niche. An understanding of the mechanisms underlying both embryonic and adult stem cell functions has been greatly enhanced by the recent development of several high-throughput technologies: microfabricated platforms, including cellular microarrays, to investigate the combinatorial effects of microenvironmental stimuli and large-scale screens utilizing small molecules and short interfering RNAs to identify crucial genetic and signaling elements. Furthermore, the integration of these systems with other versatile platforms, such as microfluidics and lentiviral microarrays, will continue to enable the detailed elucidation of stem cell processes, and thus, greatly contribute to the development of stem cell based therapies.

Figure 1

Stem cell fate and function are regulated by a combination of intrinsic programs and signals from the microenvironment. Intrinsic determinants can consist of both genetic and epigenetic components. For example, the molecular mechanisms underlying embryonic stem cell pluripotency have begun to be determined, including transcriptional regulatory networks initiated by the expression of Oct4, Sox2, and Nanog, as well as the expression of Polycomb group proteins and distinct chromatin dynamics [1]. In addition, the importance of environmental signals in stem cell function has been highlighted by the identification of distinct stem cell niches in a wide range of organ systems [4•,5]. Overall, high-throughput analysis of stem cells, utilizing both controlled cellular microenvironments and perturbations of intrinsic elements, can provide substantial insight into the factors governing stem cell biology.

Figure 2

Extracellular matrix (ECM) microarray utilized to investigate embryonic stem cell differentiation towards an early hepatic lineage. (a) Alkaline phosphatase staining of day 1 ES cultures on ECM microarrays (scale bar, 1 mm). (b,c) Bright-field micrograph of selected X-gal–stained conditions after 3 d of culture in retinoic acid. Collagen I (C1) + collagen III (C3) + laminin (L) + fibronectin (Fn) (b) induced higher reporter activity (arrowheads) for Ankrd17, a fetal liver-specific gene, than was seen in cells cultured on C3 + L (c). Scale bars, 250 μm. Magnified views of reporter activity: scale bars, 50 μm. (d) Hierarchical depiction of ‘blue’ image area (pooled data from four microarrays) for each of the matrix mixtures. Error bars, s.e.m. (n = 32). The C1 + C3 + L + Fn culture condition induced –27-fold more reporter positive image area than the C3 + L cultures. (Figure adapted from [23••] with permission).

Figure 3

Differentiation of neural precursor cells on a spotted array of microenvironmental signals. (a) Following 70 h culture on an array containing laminin (Ln) alone or Ln in combination with various other indicated factors, cells were stained for markers of proliferation (BrdU, blue), glial differentiation (GFAP, red), and neuronal differentiation (TUJ1, green). (b) High-resolution imaging of multiple parameters enabled the quantification of the differentiation status of single cells. Contour plots of the probability density of cells in response to a selection of stimuli are shown. BMP-4 or BMP (bone morphogenetic protein 4), CNTF (ciliary neurotrophic factor), DLL-4 (delta-like protein 4), Jag (Jagged-1), Shh (sonic hedgehog), TGFβ (transforming growth factor β), Wnt (Wnt-3a). (Figure adapted from [26••] with permission).

Figure 4

Microwell platform for examining stem cell fates. (a) A low magnification image to illustrate the scale of the system. (b) A higher magnification image of (a) in which distinct cells can be seen. (c) A high numerical aperture (NA) image of a single well. This image was taken with an oil immersion 100x objective to demonstrate the compatibility with high NA objectives. (d) Higher magnification image of an area outlined in white in (b). Scale bars, a-b (500 μm); c-d (100 μm). (Figure adapted from [50] with permission).

Figure 5

Lentiviral microarrays for high-throughput screening of gene function. Illustrated is a microarray printed with an alternating pattern of lentiviruses expressing either GFP or short hairpin RNA specific for lamin A/C and subsequently seeded with HeLa cells. Hoechst staining of nuclei (blue), anti-lamin A/C immunofluorescence (red), and GFP fluorescence (green) are displayed as indicated. Right panel, higher magnification image of a selected region of the array (boxed). Scale bars, left (1 mm); right (200 μm). (Figure adapted from [67••] with permission).