November 15, 2014 (Vol. 34, No. 20)
Ha T. Tran research scientist The Scripps Research Institute
Rex E. Lacambacal research scientist California State University
Candace L. Lynch research scientist The Scripps Research Institute
Jeanne F. Loring, Ph.D. director The Scripps Research Institute
Inbar Friedrich Ben-Nun, Ph.D. senior scientist Lonza
Yu-Chieh Wang, Ph.D. assistant professor University of North Texas Health Science Center
Transforming Peripheral Mononucleated Blood Cells
Generating human induced pluripotent stem cells (hiPSCs) has attracted a great deal of attention due to the cells’ resulting differentiation potential and the elimination of the ethical hurdles associated with using human embryonic stem cells (hESCs). Stem cells hold significant potential for medical research, and thus researchers are working hard to devise improved protocols to be used in their reliable creation.
hiPSCs are generated by reprogramming somatic cells via transfection with distinct transcription factors. The delivery of these transcription factors can be achieved with several different methods; typically these include the use of retroviruses, the Sendai virus, a nonviral process involving synthetic mRNA or the use of an episomal vector derived from a virus.
Each of these methods has its associated benefits and limitations, but all of which would normally involve a multistep process with the use of various reagents and kits that potentially introduce inconsistencies in quality between components. It is now possible to make use of systems designed to include everything necessary for successful hiPSC generation and culture such as Lonza’s L7™ hiPSC Reprogramming and hPSC Culture System. These systems have proven to be effective even in reprogramming resistant cases where attempts to generate hiPSCs using Sendai- or retrovirus-mediated transfection had previously failed.
The L7 System makes use of an episomal vector derived from the Epstein-Barr virus without the associated viral packaging. Using Lonza’s Nucleofector Technology and the L7 System, it is possible to have feeder- and xeno-free cultures of hiPSCs, generated from peripheral blood mononucleated cells (PBMCs). These cells retain their pluripotency and are capable of subsequent differentiation, making them valuable assets for numerous lines of research. Here we examine the generation and subsequent differentiation of hiPSCs from PBMCs.
Getting Started: Reprogramming PBMCs
The PBMCs used in this procedure were cultured in a priming medium over a period of 6–8 days before being co-transfected. A vector cocktail containing the specific transcription factors was utilized in combination with the 4D-Nucleofector kit (Lonza). This is typically a very difficult task if using traditional transfection methods, but the Nucleofector technology allows for cells to be transfected consistently and efficiently, with up to a 95% transfection efficiency.
Once transfection has been completed the cells were plated on six-well plates and cultured in an optimized recovery medium for two days. In order to promote healthy cell growth, the recovery medium was mixed 1:1 with fresh L7 hPSC Media BulletKit to feed the cells at two days post-transfection, and then replaced completely at day four. This process began to yield hiPSC colonies at approximately 7–10 days after transfection (Figure 1).
Maintenance: Culturing hiPSCs
Successful culture of the transfected cells involved replacing the medium every other day with 2 mL of fresh medium until the colonies were large enough to be subcultured. Cells would normally require feeding every day with supplemented culture medium; however, the L7 hPSC Media BulletKit can help to reduce workload as it supports every-other-day feeding, affording more flexibility.
Colonies were initially passaged manually (P1) into separate 12-well coated plates (L7 hPSC Matrix). This matrix is exceedingly helpful throughout all stages of cell culture as it supports efficient attachment and high visibility as well as helping to maintain pluripotency. A humidified incubator kept at 37°C under normoxic conditions (20.9% O2; 5% CO2) is ideal to promote healthy cell growth.
Cells at P2 were manually passaged onto radiation-inactivated mouse embryonic fibroblast (MEF) feeders (L7 hPSC Passaging Solution), using nonenzymatic cell detachment and eliminating the need for mechanical manipulation, which has been known to cause karyotype issues. It would also be possible to subculture hiPSCs colonies during their expansion at P3 and later stages.
Validating the Cellular Potential
The successful reprogramming and culture of hiPSCs can be challenging in themselves, but the procedure’s success is measured by the ability of the hiPSCs to then differentiate into different cell types. This is an extremely important validation step if the differential potential of these cells is to be harnessed for further research. To assess this, hiPSCs were differentiated into germ layers by embryoid body (EB) formation. This involved harvesting cells and culturing them in ultralow attachment plates with specialized media containing nonessential amino acids for a week. EBs were then fixed, permeabilized, and stained with fluorescent biomarkers relevant to the three germ layers (Figure 2).
hiPSCs also underwent directed differentiation into a specific somatic cell type; in this case, melanocytes. To initiate the directed differentiation of hiPSCs, colonies were harvested and then cultured in suspension as aggregates for one week in specialized media containing a mix of reagents including non-essential amino acids and β-mercaptoethanol. Cell aggregates were then plated and cultured in media specifically designed to induce melanocyte differentiation (Figure 3).
The procedure outlined here made use of Lonza’s hiPSC Reprogramming and hPSC Culture L7 System and successfully reprogrammed PBMCs by episomal vector-mediated transfection. The resulting healthy cells maintained their pluripotency and suitability for subsequent nondirected differentiation (EB formation) and directed (melanocyte) differentiation.
The efficient generation of hiPSCs is an essential process in multiple areas of medical research, particularly regenerative medicine. The use of stem cells generated from somatic cells through the processes highlighted here facilitates the exploration of disease mechanisms as well as novel therapeutic targets. This system serves as a powerful tool in the generation of hiPSCs, working to streamline what has traditionally been a challenging and occasionally uncertain process.
Ha T. Tran and Candace L. Lynch are research scientists at The Scripps Research Institute, Rex E. Lacambacal is a research scientist at California State University, Jeanne F. Loring, Ph.D., is director of the Center for Regenerative Medicine at The Scripps Research Institute, Inbar Friedrich Ben-Nun, Ph.D.(firstname.lastname@example.org), is senior scientist in the cell therapy research and technology group at Lonza, and Yu-Chieh Wang, Ph.D., is assistant professor in the department of pharmaceutical sciences at University of North Texas Health Science Center.