1Stem Cell Lab, School of Psychiatry, Faculty of Medicine, The University of New South Wales, 2Siriraj Center of Excellence for Stem cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, 3Neuropsychiatric Institute, Prince of Wales Hospital
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Sidhu, K., Kim, J., Chayosumrit, M., Dean, S., Sachdev, P. Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages. J. Vis. Exp. (61), e3608, doi:10.3791/3608 (2012).
Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, >100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.
All of the procedures below are carried out using aseptic techniques inside a Class II Biosafety Cabinet. Reagents and equipments used are listed in the tables below.
1. Preparation of 1.1% Alginate (w/v)
2. Preparation of CaCl2 Precipitation Bath
3. Preparation of Decapsulating Solution
4. Preparation of Serum Replacement (SR) Medium
5. Preparation of ROCK Inhibitor (Y-27632)
6. Preparation of hESC for Encapsulation
7. Encapsulation of Cells
8. Differentiation of Encapsulated hESC to DA Neurons
The encapsulated hESC are treated with RI for 3 days prior to differentiation.
9. Decapsulation of Encapsulated hESC
10. Representative Results
The diameter of alginate microcapsules is 400-500 μm. The number of cells within the capsule was estimated by calculating the total number of cells divided by total number of capsules per run. Therefore, approximately 5.0 x 104 cells per capsule was estimated. From this, we presume that the maximum number of cells the capsule can contain is approximately 1.0 x 105. The viability of encapsulated hESC is >80% (Figure 2) as determined by using using carboxyfluorescein diacetate succinimidly ester (CFDA)/propidium iodide (PI) assay. We have optimized the conditions of hESC encapsulation by decreasing the alginate concentration from 2.2% down to 1.1% and by changing the precipitation bath from barium chloride to calcium chloride. From these conditions, we showed that only the cells which were encapsulated with 1.1% calcium alginate could survive, proliferate and form EBs in vitro1. To further optimize the condition, the effects of culturing media and RI, Y-27632 were investigated. The data as presented in Figure 2 and 3 demonstrate that RI prevented dissociation-induced apoptosis and maintained cell viability and promoted cluster formation1,6. Furthermore, the viability of encapsulated hESC cultured in hFF-CM + RI was significantly higher than other groups without RI supplementation; however this was not significantly different to encapsulated hESC cultured in SR + RI. Similarly, cell proliferation using BrdU assay increased from 25% to 75% as single cells developed into clusters (Figure 3). Apoptosis assay by TUNEL revealed that the single cells in microcapsules cultured in SR medium were apoptotic (data not shown) whereas the retrieved clusters from the hFF-CM were mostly negative for TUNEL. To a certain extent, hFF-CM supplemented with bFGF also promoted the survival and proliferation of encapsulated hESCs in the absence of Y-27632. However, treatment with Y-27632 before (2 hours) and after encapsulation (for an additional 4 days) markedly enhanced viability, proliferation and cluster formation of encapsulated hESC in 1.1% calcium alginate microcapsules.
Previously we demonstrated that encapsulated hESC can be successfully differentiated to definitive endoderm1. Here, we examined the application of cell encapsulation as a 3D platform to differentiate encapsulated hESC into DA neurons. hESC, that formed embryoid bodies (EB) in capsules were direct differentiated and on decapsulation under the conditions described showed a progressive neuronal morphology (Figure 4) after 2-3 days of culture with more than 90% viability. Gene expression analysis showed a down-regulation of pluripotent marker, OCT4 while neuroprogenitor marker, PAX6 and DA neuronal marker, TH were up-regulated after 7 days of differentiation (Figure 5A). Immunofluorescent staining revealed that differentiated hESC were PAX6-positive (>80%) but OCT4-negative at day 7. Further differentiated hESC showed TH-positive (>90%) neurons after 21 days (Figure 5B). Western blot analysis also showed an up-regulation of TH expression from day 14 (Figure 5C) while PAX6 expression was down-regulated after day 21. In comparison, the cells cultured under two-dimensional (2D) environment under similar conditions (e.g. RI pre-treatment for 2 hours and RI post-treatment for 3 days prior to differentiation) maintained a high percentage of PAX6-positive cells (>80%) throughout the differentiation period and were not as efficient in differentiating to TH-positive cells (<60%) as in 3D environment provided by encapsulation (Figure 5A and C).
Several studies using mouse embryonic stem cells and hESC have demonstrated the benefits of 3D culture system in biomaterials and tissue engineering2,3. We used calcium alginate microcapsules as a suitable 3D platform to study hESC propagation and differentiation in comparison to barium alginate since hESC showed significant higher viability when encapsulated in calcium alginate than barium alginate. This culture system also allows a high-density cell culture and exchange of nutrients and oxygen across the membrane7. Spontaneous differentiation within the capsules has been previously observed and it is assumed that as undifferentiated hESC continue to proliferate within the capsules, the cells would eventually breakout of the capsules and form teratoma1. Another clinical application of encapsulation is to provide immune protection of transplanted cells from the host recipient. It is anticipated that transplantation of hESC and their derivatives may lead to immunological rejection since the low level of expression of MHC class I antigens of undifferentiated hESC is increased after differentiation8. It has also been documented that transplantation usually utilizes higher concentrations of alginate in order to circumvent the immune rejection process2,9. Our study uses a lower concentration of alginate (1.1%) which is more suitable for the in vitro culture and differentiation of hESC. It is yet to be determined whether the lower concentration of alginate we have used would illicit a similar immune response as previously reports as well as maintaining cell viability should these encapsulated hESC be transplanted in an immunocompetent host.
Our optimized encapsulation protocol for encapsulating hESC produces capsules size of 400-500 μm diameter. Capsules which are smaller than 400 μm tend to have fewer cells while larger capsules (>500 μm) result in an overpopulation of cells. hESC encapsulation requires a single cell formation, which also promotes cell apoptosis6. We have shown here that encapsulated hESC can continue to survive, proliferate and form EB. This is enhanced by pre-treating the hESC with RI prior to encapsulation, resulting in >80% hESC being viable. Thus, we have established a model to culture hESC in 3D culture conditions and have extended these studies for directed differentiation into DA neurons. Although cell encapsulation technique has been widely well-known for cell culturing and endodermal differentiation, neural differentiation under these conditions has not been studied thoroughly10,11. We have shown here that there is an increased expression of PAX6 and TH using gene and protein expression analyses after 7 days in comparison to 2D differentiation system, suggesting that the 3D environment promotes better DA neuronal lineage from pluripotent state. However, further analyses such as dopamine secretion test and transplantation assay are required to fully characterize the differentiated cells. Generating robust functional DA neurons efficiently is an essential requirement if cell therapy for Parkinson's disease is to become a reality. Our 3D platform as proposed about co-culturing with DA neural inducing cells, PA6 cells and high-density cell culture system of DA neuronal differentiated hESC via encapsulation is an effort towards that direction.
We have nothing to disclose.
This work is supported by NHMRC Program Grant # 568969 (PSS) and Faculty of Medicine, University of New South Wales, Stem Cell Initiative (KSS).
|Alginate (Pronova UP MVG)||NovaMatrix||4200101||high glucuronic acid content ≥60%, viscosity >200 mPa s, and endotoxin <100 EU/g|
|0.9% NaCl||Baxter Internationl Inc.||AHF7123|
|Type J1 bead generator||Nisco engineering Inc||SPA-0447|
|Multi-Phaser syringe pump||New Era||Model NE-1000|
|Ezi-Flow Medical Flowmeter||Gascon Systems||G0149|
|Y-27632||Merck & Co., Inc.||688000|
|Human Serum Albumin||Sigma-Aldrich||A4327-1G|
|14G x 2” I.V. catheter||Terumo Medical Corp.||SR-OX1451C|
|Knockout-DMEM||Invitrogen||10829-018||For SR medium (basal)|
|GlutaMAX -I||Invitrogen||35050-061||For SR medium (2 mM)|
|Knockout Serum Replacement||Invitrogen||10828-028||For SR medium (20%)|
|Penicillin-Streptomycin||Invitrogen||15070063||For SR medium (2.5 U/ml)|
|Insulin-Transferrin-Selenium (ITS)||Invitrogen||41400045||For SR medium (1x)|
|β-Mercaptoethanol||Invitrogen||21985-023||For SR medium (0.1 mM)|
|MEM NEAA Solution||Invitrogen||11140-050||For SR medium (5 mM)|
|Glasgow Minimum Essential Medium||Invitrogen||11710035||For DA neural differentiation medium (basal)|
|Knockout Serum Replacement||Invitrogen||10828-028||For DA neural differentiation medium (10%)|
|Sodium pyruvate||Invitrogen||11360070||For DA neural differentiation medium (1 mM)|
|MEM NEAA Solution||Invitrogen||11140-050||For DA neural differentiation medium (0.1 mM)|
|β-Mercaptoethanol||Invitrogen||21985-023||For DA neural differentiation medium (0.1 mM)|
|Sonic hedgehog (SHH)||R&D Systems||1314-SH-025/CF||For DA neural differentiation (100 ng/ml)|
|Fibroblast growth factor 8a (FGF8a)||R&D Systems||4745-F8-050||For DA neural differentiation (100 ng/ml)|