Engineering Three-Dimensional Stem Cell Morphogenesis for the Development of Tissue Models and Scalable Regenerative Therapeutics.
ABSTRACT The physiochemical stem cell microenvironment regulates the delicate balance between self-renewal and differentiation. The three-dimensional assembly of stem cells facilitates cellular interactions that promote morphogenesis, analogous to the multicellular, heterotypic tissue organization that accompanies embryogenesis. Therefore, expansion and differentiation of stem cells as multicellular aggregates provides a controlled platform for studying the biological and engineering principles underlying spatiotemporal morphogenesis and tissue patterning. Moreover, three-dimensional stem cell cultures are amenable to translational screening applications and therapies, which underscores the broad utility of scalable suspension cultures across laboratory and clinical scales. In this review, we discuss stem cell morphogenesis in the context of fundamental biophysical principles, including the three-dimensional modulation of adhesions, mechanics, and molecular transport and highlight the opportunities to employ stem cell spheroids for tissue modeling, bioprocessing, and regenerative therapies.
- [Show abstract] [Hide abstract]
ABSTRACT: In previous studies, fluorapatite (FA) crystal-coated surfaces have been shown to stimulate the differentiation and mineralization of human dental pulp stem cells (DPSCs) in two-dimensional cell culture. However, whether the FA surface can recapitulate these properties in three-dimensional culture is still unknown. This study examined the differences in behavior of human DPSCs cultured on electrospun polycaprolactone (PCL) NanoECM nanofibers with or without the FA crystals. Under near-physiologic conditions, the FA crystals were synthesized on the PCL nanofiber scaffolds. The FA crystals were evenly distributed on the scaffolds. DPSCs were cultured on the PCL+FA or the PCL scaffolds for up to 28 days. Scanning electron microscope images showed that DPSCs attached well to both scaffolds after the initial seeding. However, it appeared that more multicellular aggregates formed on the PCL+FA scaffolds. After 14 days, the cell proliferation on the PCL+FA was slower than that on the PCL-only scaffolds. Interestingly, even without any induction of mineralization, from day 7, the upregulation of several pro-osteogenic molecules (dmp1, dspp, runx2, ocn, spp1, col1a1) was detected in cells seeded on the PCL+FA scaffolds. A significant increase in alkaline phosphatase activity was also seen on FA-coated scaffolds compared with the PCL-only scaffolds at days 14 and 21. At the protein level, osteocalcin expression was induced only in the DPSCs on the PCL+FA surfaces at day 21 and then significantly enhanced at day 28. A similar pattern was observed in those specimens stained with Alizarin red and Von Kossa after 21 and 28 days. These data suggest that the incorporation of FA crystals within the three-dimensional PCL nanofiber scaffolds provided a favorable extracellular matrix microenvironment for the growth, differentiation, and mineralization of human DPSCs. This FA-modified PCL nanofiber scaffold shows promising potential for future bone, dental, and orthopedic regenerative applications.Journal of Dental Research 08/2014; · 4.14 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Cardiomyocytes derived from human pluripotent stem cells (hPSCs) are a promising cell source for regenerative medicine, disease modeling, and drug discovery, all of which require enriched cardiomyocytes, ideally ones with mature phenotypes. However, current methods are typically performed in 2D environments that produce immature cardiomyocytes within heterogeneous populations. Here, we generated 3D aggregates of cardiomyocytes (cardiospheres) from 2D differentiation cultures of hPSCs using microscale technology and rotary orbital suspension culture. Nearly 100% of the cardiospheres showed spontaneous contractility and synchronous intracellular calcium transients. Strikingly, from starting heterogeneous populations containing $10%–40% cardiomyocytes, the cell population within the generated cardiospheres featured $80%–100% cardiomyocytes, corresponding to an enrichment factor of up to 7-fold. Furthermore, cardiomyocytes from cardiospheres exhibited enhanced structural maturation in comparison with those from a parallel 2D culture. Thus, generation of cardiospheres represents a simple and robust method for enrichment of cardiomyocytes in microtissues that have the potential use in regenerative medicine as well as other applications.Stem cell reports. 07/2014; 3(2).
- [Show abstract] [Hide abstract]
ABSTRACT: Human pluripotent stem cell (hPSC)-derived endothelial cells and their progenitors may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. Here, we report a method to efficiently produce endothelial cells from hPSCs via GSK3 inhibition and culture in defined media to direct hPSC differentiation to CD34(+)CD31(+) endothelial progenitors. Exogenous vascular endothelial growth factor (VEGF) treatment was dispensable, and endothelial progenitor differentiation was ?-catenin dependent. Furthermore, by clonal analysis, we showed that CD34(+)CD31(+)CD117(+)TIE-2(+) endothelial progenitors were multipotent, capable of differentiating into calponin-expressing smooth muscle cells and CD31(+)CD144(+)vWF(+)I-CAM1(+) endothelial cells. These endothelial cells were capable of 20 population doublings, formed tube-like structures, imported acetylated low-density lipoprotein, and maintained a dynamic barrier function. This study provides a rapid and efficient method for production of hPSC-derived endothelial progenitors and endothelial cells and identifies WNT/?-catenin signaling as a primary regulator for generating vascular cells from hPSCs.Stem cell reports. 11/2014; 3(5):804-16.
Bio-Medical Materials and Engineering 18 (2008) 179–181
Engineering the 3D microenvironment of
embryonic stem cells undergoing
Todd C. McDevitt∗, Richard L. Carpenedo, Carolyn Y. Sargent, Geoffrey Y. Berguig,
Ross A. Marklein and Scott Seaman
The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology,
Emory University, Atlanta, GA, USA
The Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology,
Atlanta, GA, USA
Embryonic stem (ES) cells are pluripotent cells that may be used for regenerative medicine treatments
somatic cell types. In order to translate the potential of ES cells into viable cellular therapies, enhanced
methods for controlled differentiation ex vivo prior to transplantation need to be developed. Stem cells
are responsive to a number of different environmental cues capable of affecting cell fate decisions, thus
engineering approaches to more precisely regulate the extracellular microenvironment of stem cells may
enhance the efficiency, yield and homogeneity of differentiating ES cell derivatives for regenerative cell
ES cells are commonly induced to spontaneously differentiate in vitro by aggregation in suspension
conditions into “embryoid bodies” (EBs). EB differentiation recapitulates the sequence of molecular and
cellular events that normally occur during the pre-implantation stages of development to yield primitive
ectoderm, endoderm and mesoderm cell types. Within individual EBs, cellular differentiation is inher-
ently heterogeneous and can vary significantly between different EBs, thus often limiting the ability to
consistently achieve directed differentiation of ES cells into defined cell phenotypes using batch cul-
ture methods. The objective of our studies is to improve the uniformity of ES cell differentiation by
simultaneously controlling the homogeneity of EB formation and differentiation in suspension culture
conditions, as well as regulating morphogenic factor presentation to ES cells within EB environments.
Our results suggest that these goals can be accomplished via the use of novel rotary orbital suspension
methods for EB culture and integration of degradable microparticles within EBs to spatiotemporally
control the presentation of differentiation factors to cells locally.
*Address for correspondence: T.C. McDevitt, The Wallace H. Coulter Department of Biomedical Engineering, Georgia
Institute of Technology, Emory University, Atlanta, GA, USA. E-mail: email@example.com.
0959-2989/08/$17.00 © 2008 – IOS Press and the authors. All rights reserved