September 1, 2011 (Vol. 31, No. 15)
Combining Small Molecules and a Polycistronic Vector for Disease Modeling and Toxicity Screening
The ability to “reprogram” differentiated adult cells to a state that resembles embryonic stem cells has created wide-ranging opportunities for development of relevant in vitro disease models and patient-specific cell-replenishment therapies.
Initial efforts to generate human induced pluripotent stem cells (iPS cells) required simultaneous co-infection of cells with four separate retroviral expression vectors (Oct-4, Klf4, Sox-2, and c-Myc). Each vector carried one transcription factor, which resulted in a high number of genomic integrations.
Alternative approaches to iPS generation have included use of plasmids and nonintegrating adenovirus vectors to deliver the transcription factors. The rates at which cells convert to pluripotency using these methods, however, are far lower than those obtained using retroviral vectors.
Generation of human and mouse iPS cells is now routinely accomplished using a single, excisable polycistronic lentiviral vector that delivers all four Yamanaka transcription factors. EMD Millipore’s STEMCCA™ lentiviral-based reprogramming yields cells that form multilayered tightly packed colonies with well-defined borders. These cells stain positive for alkaline phosphatase, express embryonic stem cell markers, form embryoid bodies, and differentiate into all three germ layers.
Use of a single vector significantly reduces the number of viral integrations required—in some cases, iPS clones possessing only a single viral integrant can be isolated.
Even with the use of a single vector, however, reprogramming human somatic cells remains a highly inefficient and time-consuming process. Small molecules targeting specific signaling pathways are being investigated for their ability to enhance reprogramming and/or replace the transcription factors required for reprogramming.
In this study, chemical compounds were screened for their effects on increasing the ratio of fully reprogrammed SSEA4+TRA-1-60+ Hoechst dim iPS cells versus reprogramming intermediates, increasing colony numbers, and reducing the time to establishment of full reprogrammed iPS cell colonies.
Improving Reprogramming Efficiency
Human foreskin fibroblasts were seeded at a density of 10,000/well and transduced with the Human STEMCCA Constitutive Polycistronic Lentivirus Reprogramming Kit (EMD Millipore). Various combinations of small molecules involved in TGF, Wnt, and MAPK signaling pathways along with epigenetic modifiers were screened.
A total of 25 small molecule boost cocktails were evaluated; boost cocktails were added at day six after replating the fibroblasts onto a feeder layout of inactive mouse embryonic fibroblasts. Reprogramming without chemical treatment was used as a control for all experiments.
Small molecule boost cocktails were identified that increased the number of iPS colonies by up to two- to threefold (Figure 1). The colonies possessed the distinctive 2-D morphology that is reminiscent of human embryonic stem cells and could be easily passaged in contrast to the untreated control that exhibited 3-D morphology and were difficult to passage.
In addition, some treatments yielded colonies with proliferation kinetics similar to human embryonic stem cells starting at the first passage rather than passage three as seen with untreated controls.
Chemically treated human iPS cells possessed fast proliferation kinetics; early passages from P0 to P3 were shortened to five to six days per passage period—a timeframe that is similar to the proliferation rate of normal human embryonic stem cells.
Treatment 2 was selected on the basis that it significantly improved both the quality of colonies formed and the efficiency of reprogramming. Treatment 2 is herein referred to as Human iPS Cell Boost Supplement.
Figure 1. Effect of small molecule combinations on colony morphology, number of colonies generated, ease of passaging, and relative proliferation rate as measured by days to passaging: Human foreskin fibroblasts were reprogrammed with mouse STEMCCA lentivirus. Treatment 2 was selected for further characterization.
In the presence of the Human iPS Cell Boost Supplement, the number of colonies formed increased threefold (Figure 2A) when used in combination with the mouse STEMCCA lentivirus kits and 15-fold when used in combination with the human STEMCCA lentivirus kits. Colonies stained positive for both human ESC markers, SSEA-4 (Figure 2F), and TRA-1-60 (Figure 2G). TRA-1-60+ colonies were not observed in the untreated control cultures (Figure 2D).
Figure 2. Addition of Human iPS Cell Boost Supplement dramatically increased the efficiency of colony formation (A) and shortened the time to establishment of full reprogrammed human iPS clones (E, F, G). Human iPS Cell Boost Supplement enhanced colony formation by two- to threefold when used in combination with the mouse STEMCCA lentivirus kits and 15-fold when used in combination with the human STEMCCA lentivirus kits (A). Four independent experiments (MOI=200) were performed using mouse STEMCCA lentivirus kit while two experiments were performed (MOI=10) using human STEMCCA lentivirus kits. Reprogramming without chemical treatment was used as a control for all experiments. P0 human iPS colonies generated from FibroGRO™ Xeno-free Human Foreskin Fibroblasts reprogrammed with mouse STEMCCA lentivirus in the presence of the Human iPS Cell Boost Supplement exhibited larger colony sizes, a flat 2-D morphology (E), and were SSEA-4-positive (F) and TRA-1-60-positive (G). This is in marked contrast to untreated control where the colonies were smaller, were 3-D in morphology (B), and were SSEA-4 positive (C) but TRA-1-60-negative (D) at similar timepoints.
Reprogramming with small molecules and the polycistronic vector was further validated on multiple human fibroblast cell lines in both feeder- and serum-free conditions. Chemical treatment improved reprogramming in StemPro® medium on Geltrex™-coated plates (Life Technologies) and in mTeSR®1 medium (Stemcell Technologies) on BD Matrigel™-coated plates (BD Biosciences), although a decreased kinetics of reprogramming was observed when compared to culture on feeder cells (Figure 3).
The enhancement of reprogramming by chemical treatment has also been demonstrated using other human fibroblast cell lines.
Figure 3. Chemical treatment improves reprogramming effectively in feeder-free and serum-free conditions.
Conclusion
Use of small molecules that modulate key signaling pathways and epigenetic modifiers enables dramatic improvement in the quality and quantity of human iPS colonies generated.
Human iPS Cell Boost Supplement enhanced colony formation by two- to threefold when used in combination with the mouse STEMCCA lentivirus kits and 15-fold when used in combination with the human STEMCCA lentivirus kits.
Human iPS colonies generated in the presence of Human iPS Cell Boost Supplement could be readily expanded for over 30 passages. Human iPS cells displayed the morphology characteristic of human ESCs, had a normal karyotype, and stained positive for pluripotent markers.
Use of the polycistronic lentiviral expression cassette in combination with small molecules provides a convenient platform for screening small molecules that enhance reprogramming efficiency with the eventual goal of generating iPS cells without genetic modification of the genome.
iPS cells have the potential to reshape the research and clinical landscape. Use of these cells for disease modeling and drug and toxicity screening can help overcome the limitations of current methods, enable construction of human models of complex diseases, and reveal important insights that can lead to a more personalized approach to medicine. Methods to improve the efficiency and kinetics of reprogramming human somatic cells into iPS cells will accelerate the application of these cells in both the research and clinical settings.
Min Lu ([email protected]) and Cristina Moore are research scientists, Amy Noble is product manager, and Vi Chu is R&D manager, all in stem cells/cell biology at EMD Millipore.
