Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells

Highlights

Summary

Introduction
Endothelial cells (ECs) are major components of blood vessels (Carmeliet, 2001, Richards et al., 2010). They are of great value for disease modeling, drug screening, cell therapy, and tissue engineering (Heo et al., 2014, Huang et al., 2013, Kang et al., 2013, Leung et al., 2016, Medina et al., 2010, Moubarik et al., 2011, Patsch et al., 2015, Schwarz et al., 2012, Stroncek et al., 2012). However, obtaining large numbers of primary ECs for those applications, in particular for clinical applications (Arici et al., 2015, Chao et al., 2014, den Dekker et al., 2011, Granton et al., 2015, Matoba et al., 2008), is still challenging due to their limited proliferation capacity and phenotype changes during the in vitro culture (van Beijnum et al., 2008, de Carvalho et al., 2015, Gui et al., 2009, Gumbleton and Audus, 2001, Hayflick, 1965, Augustin-Voss et al., 1993). Human pluripotent stem cells (hPSCs) provide a potential solution to this challenge (Levenberg et al., 2007). hPSCs, including human embryonic stem cells (hESCs) (Thomson et al., 1998) and induced pluripotent stem cells (iPSCs) (Takahashi et al., 2007, Yu et al., 2007), have unlimited proliferation capacity and can be efficiently differentiated into ECs through 3D embryonic body (EB)-based (Condorelli et al., 2001, James et al., 2010, Levenberg et al., 2002, Levenberg et al., 2007, Li et al., 2009a, Li et al., 2009b, Nourse et al., 2010) or 2D monolayer culture-based protocols (Cao et al., 2013, Kane et al., 2010, Palpant et al., 2016, Patsch et al., 2015, Vodyanik et al., 2005). In addition, cells derived from patient-specific iPSCs have the patient’s genetic information and can model many human diseases. Further, they induce minimal immune response in vivo (Lalit et al., 2014). These hPSC-derived ECs have the potential to provide unlimited cell sources for the applications.
While making small-scale hPSC-derived ECs in laboratories can be readily done (Giacomelli et al., 2017, Lian et al., 2014, Orlova et al., 2014, Palpant et al., 2016, Zhang et al., 2017a), generating or manufacturing large numbers of ECs from hPSCs has not been achieved. Current 2D culture methods, in which cells are cultured as adherent cells on 2D surfaces (e.g., cell culturing flasks), are labor, time, and cost intensive, and not suitable for culturing cells on a large scale (Jenkins and Farid, 2015, Kropp et al., 2017). 3D suspension culture methods (e.g., using stirred-tank bioreactors), in which cells are suspended in an agitated culture medium, have been considered a potential solution for scaling up the cell production (Jenkins and Farid, 2015, Kropp et al., 2017, Lei and Schaffer, 2013). However, recent research has shown that culturing cells on a large scale with 3D suspension cultures is also very challenging (Lei et al., 2014, Serra et al., 2012, Steiner et al., 2010, Wurm, 2004). hPSCs in 3D suspension cultures frequently aggregate to form large cell agglomerates (Kropp et al., 2017). The mass transport to cells located at the center of large agglomerates (e.g., >400 μm diameter) becomes difficult, leading to slow cell growth, cell death, and uncontrolled differentiation (Kropp et al., 2017). While agitating the culture can reduce cell agglomeration, it also generates hydrodynamic stresses, which are adverse to the cell’s physiology (Fridley et al., 2012, Kinney et al., 2011, Kropp et al., 2017). As a result, 3D suspension culturing has significant cell death, low cell growth, and low volumetric yield (Lei and Schaffer, 2013). For instance, hPSCs typically expand 4-fold in 4 days to yield around 1.0 × 106 to 2.0 × 106 cells/mL, which occupy ∼0.4% of the bioreactor volume (Lei et al., 2014, Serra et al., 2012, Steiner et al., 2010, Wurm, 2004).
To address the challenge, we previously developed a scalable, efficient, and current Good Manufacturing Practice (cGMP)-compliant method for expanding hPSCs (Lei and Schaffer, 2013, Li et al., 2016, Lin et al., 2017). The method, which was successfully repeated in this study (Figures 1 and S2), uses a 3D thermoreversible hydrogel (Mebiol Gel) as the scaffold. Single hPSCs are first suspended in a liquid PNIPAAm-PEG polymer solution at low temperature (e.g., 4°C). Upon heating to 20°C–37°C, the polymer solution forms an elastic hydrogel matrix, resulting in single hPSCs encapsulated in the hydrogel matrix. After culturing for about 4–5 days, these single hPSCs clonally grow into spherical cell aggregates (spheroids) with very uniform size (Figures 1B, S2A, and S2D). The hydrogel can be quickly liquefied through cooling to ∼4°C to harvest the cells for the next passage (Figure 1A). The hydrogel scaffold protects cells from hydrodynamic stresses in the culture vessel and prevents cells from excessive agglomeration, leading to high culture efficiency. For instance, the hydrogel scaffold enables long-term, serial expansion of hPSCs with a high cell viability (e.g., >90%, Figures 1D, S2C, and S2F), growth rate (e.g., 20-fold/5days, Figure 1E), yield (e.g., 2.0 × 107 cells/mL, Figure 1F), and purity (>99%, Figure 1C, S2B, and S2E), all of which offer considerable improvements over 3D suspension cultures (Lei and Schaffer, 2013, Li et al., 2016, Lin et al., 2017). We hypothesize that hPSCs can also be differentiated into ECs in this culture system. In this paper, we successfully tested the hypothesis. Together, we developed a scalable bioprocess for making high-quality ECs with high volumetric yield, high viability, and high purity (>80%).

Results

Differentiation of hPSCs into ECs in 2D Adherent Cultures
We used H9 hESCs, iPSCs reprogrammed from human dermal fibroblasts (i.e., Fib-iPSCs), and iPSCs reprogrammed from mesenchymal stem cells (i.e., MSC-iPSCs) (Park et al., 2008), for this study. All formed compact colonies when cultured on Matrigel-coated plates in the chemical-defined Essential 8 (E8) medium (Figures S1A, S1E, and S1I). They expressed pluripotency makers OCT3/4 and NANOG (Figures S1B, S1F, and S1J), and could be differentiated into all three germ layer cells (e.g., NESTIN+ ectodermal, α-SMA+ mesodermal, and HNF-3β+ endodermal cells) in EB assay (Figures S1C, S1G, and S1K). They also formed teratomas containing all three germ layer tissues in immunodeficient mice (Figures S1D, S1H, and S1L).

Patsch et al. (2015) recently reported a protocol that could efficiently generate ECs from hPSCs in 6 days in 2D cultures. This protocol is simple and quick, and thus it is very appealing for making high-quantity ECs. We successfully repeated this protocol with our H9s and iPSCs (Figure S3). The produced ECs had the typical EC cobblestone morphology (Figures S3B and S3H). Immunostaining showed that the majority of these cells expressed the EC markers PECAM1 (or CD31) and VE-Cadherin (or CD144) (Figures S3C and S3I). Flow cytometry analysis showed that about 80% cells were positive for the two markers (Figures S3D and S3J). A small fraction of produced cells was positive for SM22A and CD140b, markers for smooth muscle cells (Figures S3E, S3F, and S3K). We did not detect any undifferentiated OCT3/4+ and NANOG+ hPSCs (Figures S3G and S3L). H9s and iPSCs had similar outcomes (Figure S3). Our results were very similar to these reported by Patsch and Cowan, indicating the robustness of the differentiation protocol (Patsch et al., 2015). We termed ECs made in 2D culturing as 2D-ECs.

Differentiation of hPSCs into ECs in 3D Thermoreversible PNIPAAm-PEG Hydrogels
We then applied the protocol to differentiate hPSCs in the 3D thermoreversible hydrogels (Figure 2A). Single hPSCs were encapsulated into the gel and expanded for 5 days to generated hPSC spheroids with a diameter of around 150 μm. Differentiation was initiated on day 0 by switching the expansion medium to the differentiation medium (Figure 2B). Live/dead cell staining showed that the majority of cells on day 5 were live (Figure 2C). Immunostaining and confocal imaging showed that the majority of cells in the day 5 spheroids were positive for EC markers PECAM1 and VE-Cadherin (Figure 2D). ECs were uniformly distributed, and no cysts were found in the spheroids, indicating no or little cell death in the spheroids. Flow cytometry analysis found that about 84% of the cells were PECAM1+ and VE-Cadherin+ (Figure 2E). About 1.6 × 107cells and 2.0 × 107 cells were produced in each milliliter of hydrogel on day 0 and 5, respectively (Figure 2F). Thus, about 20 cells were generated from one input hPSC on day 5. When the day 5 EC spheroids were dissociated into single cells and plated on Matrigel-coated plates at high density, they formed tight cell-cell interactions (Figure 2G). Immunostaining detected small numbers of SM22A+ cells (Figure 2H), and flow cytometry analysis found that about 13.8% of cells were CD140b+ (Figure 2I). No undifferentiated OCT3/4+ and NANOG+ hPSCs were detected (Figure 2J). The majority of cells were PECAM1+/VE-Cadherin+ (Figure 2K). Fib-iPSCs and MSC-iPSCs had similar outcomes (Figures S4 and S6). We found the differentiation efficiencies in the 3D hydrogel and the conventional 2D culture were very close (Figures 2 and S3). We termed ECs made in the hydrogel and that had not been cultured on any 2D surfaces as 3D-ECs.