An alternative strategy to the above conventional lineage conversion method was recently developed that employs transient overexpression of iPSC-TFs in conjunction with lineage specific soluble signals to reprogram somatic cells into diverse lineage-specific cell types without entering the pluripotent state.
This paradigm came from the study of iPSC reprogramming, in which iPSCs are generated through a lengthy and inefficient process with stochastic events. Only a few cells finally become pluripotent but many cells “land” in other non-pluripotent states. We hypothesized that initial overexpression of iPSC-TFs may induce an “epigenetic activation” (e.g., destabilizing and erasing starting cell’s epigenetic state, and enabling more permissive states for genes in other lineages), and that temporally controlled expression of exogenous iPSC-TFs could interact with TFs downstream of lineage-specific signals (i.e., culture conditions) to initiate transcriptional cascades and establish the reprogrammed cell’s epigenetic landscape.
With this rationale, we found that mouse fibroblasts could be converted into cardiomyocytes through temporally restricted expression of Oct4, Klf4, Sox2 (as short as 4 days) followed by BMP4 treatment, without entering the pluripotent state [70••
]. During this process, a JAK inhibitor (JI1) was added into the culture condition to block iPSC formation and increase the lineage conversion efficiency. Compared with previous methods of cardiac lineage conversion by overexpression of cardiac specific transcription factors, it is more efficient and less time consuming. The spontaneously beating cell patches were observed as early as 11 days post induction. Importantly, those mature (cTnT+) cardiomyocytes were generated through cardiac precursor (Flk-1+ and Isl1+) populations using this method. Due to issues of the required number of cells for transplantation and cell survivability in vivo
, proliferating lineage-specific progenitor cells could be more promising for future regenerative therapies than terminally differentiated cells. Through a similar strategy, neural progenitor cells (NPCs) can also be reprogrammed from fibroblasts by transient expression of the iPSC reprogramming factors followed by treatment with corresponding cell lineage-specific growth factors and small molecules [71
]. Remarkably, it was demonstrated that the induced NPCs could be isolated, expanded in vitro
, and then further differentiated into functional neuronal and glial cell types. Recently, two other groups also successfully derived mouse neural stem cells (NSCs) using transduction of Sox2, Klf4, c-myc, together with either two other transcription factors, Brn4 and E47, or transient induction of Oct4 [72
]. Importantly, those induced NSCs can differentiate into neurons, astrocytes and oligodendrocytes, and maintain their differentiation potential over many passages.
Compared with conventional lineage conversion, this new method has several advantages. Expression of a single set of TFs could be better optimized for different cell lineages. In addition, transient expression of TFs might be more amenable to non-integrating or non-genetic methods for inducing lineage conversion, such as using miRNA, mRNA and small molecules. Furthermore, generating multipotent progenitor populations could be more useful than reprogramming directly into mature non-proliferative cells, as is common with conventional lineage reprogramming, for many applications. The mechanisms of conventional and iPSC-TF mediated lineage conversion are quite different. In the conventional paradigm, a cell is forced to adopt another fate by master transcription factors of the target cell type, whereas in iPSC-TF mediated lineage conversion, the original cell fate is destabilized and the cells are partially “dedifferentiated” by the iPSC-TFs, enabling their differentiation to be directed and patterned with soluble factors. Because the trans-differentiation of the iPSC-TF initiated cells follows, in part, natural development, this method can benefit from the field of directed differentiation. It will remain a question which paradigm will be more useful for various applications in terms of efficiency and fidelity until more thorough investigations have been performed.
Collectively, the four conventional iPSC factors not only induce reprogramming to iPSCs, but also are capable of mediating direct cell fate switching between somatic cells. Changing the duration of transgene expression and culture conditions may allow establishing a transient, plastic state and effectively serve as a cellular platform for lineage conversion toward various lineages. With continued advances in iPSC technology, many small molecules that have been identified to enhance iPSC reprogramming may also have a positive role in lineage-specific programming, especially in iPSC-reprogramming-factors-induced lineage conversion. For example, small molecules that modulate epigenetic processes may promote erasure of the original epigenetic state of initial cells and accelerate the maturation and increase the function of transdifferentiated cells.