Cellular reprogramming represents the biological equivalent of spinning gold from straw: the generation of one therapeutically-desired cell type from another more abundant, and less useful, starting cell type. Following reprogramming, we can incorporate the resultant cells into new and effective patient-specific regenerative strategies.
Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) generates a pluripotent population that can be amplified and differentiated, while direct reprogramming of somatic cells forgoes the pluripotency stage and directly produces the desired differentiated cell type. Both reprogramming strategies generally employ transduction of viral vectors expressing a set of lineage-specific transcription factors which aim to jump-start a different cellular program within the target cell.
However, this can be a complex and expensive process, with the risk of insertional mutagenesis. To make the process easier and cheaper, researchers have attempted to remove the transcription factors and their vectors completely and pharmacologically replace them with small molecules inhibitors of signaling pathways/epigenetic modifications and growth factors. While this has been a successful strategy for iPSC generation, small molecule control of direct reprogramming has proven a more arduous task.
Until now that is, as a double-shot of research from the Rumpelstiltskin of chemical reprogramming, Sheng Ding (Gladstone, UCSF), has now described direct “spinning” of human and mouse cells (straw) into cells resembling cardiomyocytes (ciCMs) and neural stem cells (ciNSLCs) using small molecule cocktails (GOLD!).
Chemically-induced Heartbeats
The first of these two papers from Zhang et al appeared in Science and aimed to directly reprogram human fibroblasts into cardiomyocytes (heart muscle cells that control heart contraction), with the eventual aim of using such cells in regenerative therapies for human heart problems (i.e., heart attacks). The authors of the study initially tested 89 small molecule compounds, and from this list, refined their cocktail recipe to a mere 9 compounds (9C) – simple enough to make anyone’s heart beat faster!
Let’s have a deeper look at the recipe, which included various signal pathway modulators and chromatin modifiers in the hope of opening the closed chromatin conformation of the fibroblasts and “kick-starting” the cardiomyocyte transcriptional program.
- CHIR99021 – GSK3 inhibitor that activates the Wnt pathway
- A83-01 – TGF-beta receptor inhibitor that inhibits TGF-beta signaling
- BIX01294 – GLP and G9a histone lysine methyltransferase inhibitor
- AS8351 – Histone demethylase inhibitor
- SC1 – ERK inhibitor
- Y27632 – ROCK inhibitor
- OAC2 – Reprogramming booster
- SU16F and JNJ10198409 – PDGF receptor inhibitors
Treatment with this cocktail for 20-30 days generated highly homogenous and functional CMs (ciCMs) that closely resembled CMs derived from human pluripotent stem cells (hPSCs) at the electrophysiological, epigenetic, and transcriptomic levels. Excitingly, the group went on to demonstrate the regenerative potential of their ciCMs by transplanting 9C-treated fibroblasts directly into the mouse heart, where the efficiently converted to ciCMs.
Pharm-ing Neural Stem Cells
The next paper from Cao et al appeared in Cell Stem Cell and used the same strategy in an attempt to produce large amounts of functional neural stem cells (NSCs). This study used mouse embryonic fibroblasts as their starting material, and again, they produced a refined small molecule cocktail laden with chromatin modifiers, signal pathway modulators, and growth factors, which they hoped would promote the emergence of the neural lineage.
Their new recipe, guaranteed to leave your taste bud nerve endings ready for more, included:
- CHIR99021 – GSK3 inhibitor that activates the Wnt pathway
- LDN193189 – Inhibitor of BMP type I receptor ALK2/3 that prevents Smad phosphorylation
- A83-01 – TGF-beta receptor inhibitor that inhibits TGF-beta signaling
- Retinoic Acid – Enhances neural differentiation
- Hh – Smo agonist that can promote neural differentiation
- RG108 – DNA methyltransferase inhibitor
- Parnate – Histone demethylase inhibitor
- SMER28 – Autophagy modulator
Similar to the cardiomyocyte study, this cocktail generated highly homogenous neural stem cell-like cells (ciNSLCs) in around 10 days. ciNSLCs resembled primary NSCs with regards to marker expression and abilities to self-renew and differentiate in vitro and in vivo into neurons, astrocytes, and oligodendrocytes.
Safer, Cheaper, and Better Reprogramming?
The small molecule cocktails described in this study represent a new means to produce patient-specific replacement cells, with multiple advantages over transcription-factor transduction-based reprogramming protocols. There are no integration events or potentially oncogenic transcription factors, so the process is much safer. Small molecules are easier to use and deliver, allowing a previously inconceivable level of reprogramming control. Finally, the whole process is also cheaper than traditional methods and is more amenable for scale-up to produce the numbers of cells required for human therapeutic uses.
The next question is what cell will come next – hematopoietic stem cells, primordial germ cells, or pancreatic b-cells maybe? Before the VIP list for this reprogramming party grows too long, the authors do note that we still need further research into boosting reprogramming efficiency, assessing the risk of mutations arising during this mode of reprogramming, and fully understanding the potent effects of these reprogramming cocktails.
Until then, sit back, relax, and grab both your favorite cocktail and copies of these great new studies at Cell Stem Cell, April 2015, and Science, April 2015. Dry martini anyone?