Inducible and Deterministic Forward Programming of Human Pluripotent Stem Cells into Neurons, Skeletal Myocytes, and Oligodendrocytes

1Despite major efforts to develop robust protocols for scalable generation of human cell types from easily accessible and renewable sources, the differentiation of human pluripotent stem cells (hPSCs) into specific cell types often remains cumbersome, lengthy, and difficult to reproduce. Moreover, the recapitulation of developmental stages in vitro yields fetal cells that often do not reach full maturation. More recently, forced expression of lineage-specific master regulators resulting in direct reprogramming of somatic cell types has provided an efficient alternative to directed differentiation. In particular, the direct conversion of hPSCs, termed forward programming, combines the advantages of hPSC differentiation and direct cellular reprogramming, enabling scalable and rapid generation of human cell types.

2Currently available forward programming protocols are largely based on lentiviral transduction of hPSCs, which results in variegated expression or complete silencing of transgenes. Additional purification steps are usually necessary for enriching the desired cell type. Lentiviral approaches randomly insert transgenes into the genome bearing the risk of unwanted interference with the endogenous transcriptional program. Therefore, refinements to the current forward programming approaches are desirable.

3As the result of a systematic effort to optimize gene expression in hPSCs, we arrived at a robust hPSC forward programming platform by targeting all components of the Tet-ON system required for inducible expression of transcription factors into genomic safe harbor sites (GSHs). The Tet-ON system consists of two components: a constitutively expressed transcriptional activator protein responsive to doxycycline (dox) (reverse tetracycline transactivator [rtTA]), and an inducible promoter regulated by rtTA (Tet-responsive element) that drives expression of the transgene. Previous GSH-targeting strategies of the Tet-ON system relied on introducing both elements into the AAVS1 GSH of hPSCs, either separately, or together (using an all-in-one Tet-ON vector). Compared with these designs, we reasoned that targeting each of the two elements of the Tet-ON system into a different GSH would have several advantages: inducible overexpression based on dual GSH targeting would not be affected by promoter interference between the two transgenes, while homozygous GSH targeting would maximize the number of safely targeted transgene copies. Moreover, the larger cargo capacity in each of the transgenes would permit increased flexibility for transgene design, thus allowing the insertion of large reprogramming cassettes.

4Here we show that an optimized approach based on dual GSH targeting of Tet-ON-controlled transgenes results in homogeneous, controllable, and extremely high expression of inducible transgenes in hPSCs. Application of the optimized overexpression platform enabled us to develop rapid and deterministic forward programming protocols for mature human cell types.

5To optimize inducible transgene overexpression from GSHs, we generated human embryonic stem cells (hESCs) with inducible EGFP (i-EGFP) expression. Initially, we tested four different designs (Figure S1A). These comprised two all-in-one targeting constructs in which both rtTA and i-EGFP expression cassettes (third-generation Tet-ON system) were inserted into the same allele of the AAVS1 GSH locus. In these constructs, the rtTA expression was under the control of either an EF1α or CAG promoter. The other two transgene designs were based on spatial separation of the activator and responder into two distinct GSHs (Figure S1A). For this purpose, we sequentially targeted the rtTA cassette into the human ROSA26 GSH and an i-EGFP transgene into the AAVS1 GSH (Figures 1A and S1A–S1E). Robust and homogeneous inducible transgene expression was achieved only when the dual GSH approach and a CAG promoter for rtTA expression was used (Figure S1A). Importantly, the dual GSH-targeting approach was highly efficient (Table S1), and did not affect hESC self-renewal or differentiation (Figures 1H–1K).

6Using the CAG promoter-based dual GSH-targeting approach, we selected clonal lines that carried either one or two copies of each of the transgenes (Figure 1B), and observed that homozygous targeting of both elements allowed maximal inducible overexpression (Figures 1C, 1D, S1E, and S1F). Under these conditions, EGFP expression was induced homogeneously in all cells, consistent across multiple clones, and more than 50-fold higher compared with EGFP expression via the strong constitutive CAG promoter (Figures 1D, 1E, S1E, and S1F). Maximal EGFP levels were reached approximately 4 days after induction, and expression was quickly reversed upon dox withdrawal (Figure 1F). Moreover, EGFP expression could be titrated by adjusting the dose of dox (Figure 1G). I-EGFP expression was highly efficient in hESCs (Figures 1E and 1H), and during germ layer differentiation (Figures 1I–1K and S1G). There was no detectable background expression of EGFP in the absence of dox (Figures 1D, 1E, and S1G).

7Taken together, these results established that homozygous dual GSH targeting of the Tet-ON system is a powerful strategy for homogeneous and controllable expression of inducible transgenes in hPSCs and their derivatives. We will refer to this platform as “OPTi-OX” (optimized inducible overexpression).

8To test the applicability of the OPTi-OX platform for forward programming of hPSCs into mature cell types, we first chose to generate excitatory cortical neurons, as previous studies demonstrated that these can be readily derived by lentiviral overexpression of pro-neuronal transcription factors in hPSCs.

9To this end, we generated NGN2 OPTi-OX hPSCs (Figure 2A; Table S1), and treated them with dox in chemically defined neuronal culture medium. Induction of NGN2 expression (Figure S2A) resulted in downregulation of pluripotency factors and initiation of the neuronal transcriptional program (Figure 2B). Dox-treated cells extended neuronal processes as early as 3 days post induction. After 1 week, all cells displayed a neuronal morphology and expressed the pan-neuronal markers βIII-tubulin and MAP2 (Figures 2C–2E and Movie S1). At this stage, induced neurons (i-Neurons) showed strong expression of forebrain markers BRN2 and FOXG1, and of glutamatergic neuronal genes GRIA4, VGLUT1, and VGLUT2 (Figures 2B and 2F), indicative of an excitatory cortical neuronal identity of the forward-programmed cells, consistent with previous reports. Short pulses of dox treatment for 4 days or longer sufficed for complete conversion, and converted cells did not rely on continuous transgene expression (Figures S2B and S2C). Importantly, we did not observe any reduction in the efficiency of generating i-Neurons over extended culture periods of the inducible hESCs (>25 passages, Figure 2E). Finally, we confirmed the applicability of the NGN2 OPTi-OX system in hiPSCs (Figure 2G). Collectively, these results demonstrated that OPTi-OX enables robust and rapid forward programming of hPSCs into cortical neurons.

10To further explore the potential of OPTi-OX for forward programming of hPSCs, we focused on generating human skeletal myocytes. Existing protocols for the directed differentiation of skeletal myocytes from hPSCs are difficult, time consuming, and result in low and variable yields. On the other hand, myogenic transdifferentiation has been achieved by overexpressing the transcription factor MYOD1 in somatic cell types, but the ability of hPSCs to undergo MYOD1-induced forward programming is a matter of debate.

11We therefore generated MYOD1 OPTi-OX hPSCs (Figures 3A and 3B and Table S1). However, induction of MYOD1 expression following dox treatment resulted in cell death within 3–5 days, regardless of the culture medium used (data not shown). These findings demonstrated that MYOD1 overexpression alone was not sufficient to drive myogenesis in hPSCs, in agreement with the postulated existence of epigenetic barriers preventing forced myogenesis.

12Cellular reprogramming strategies can be enhanced by combining transcription factor overexpression with extracellular signaling cues. We conducted a systematic screen for pro-myogenic factors by modulating key signaling cascades that are implicated in primitive streak formation, somitogenesis, and myogenesis (Figure S3A). We found that the addition of all-trans retinoic acid (RA) in conjunction with MYOD1 overexpression was sufficient for rapid and deterministic conversion of hPSCs into myogenin and myosin heavy chain double-positive myocytes after 5 days of induction (Figures 3C and S3A). The effect of RA was concentration dependent (data not shown), and mediated at least in part through the receptor isoforms RARα and RARβ (Figures S3B and S3C).

13Following minor optimization of the culture conditions (see the Supplemental Experimental Procedures), we arrived at a protocol resulting in nearly pure induced skeletal myocytes (i-Myocytes). Reprogrammed cells developed typical spindle-like, elongated morphology, underwent extensive cell fusion, and exhibited strong and homogeneous myogenic marker expression on mRNA and protein levels (Figures 3D–3H, S3D, and S3E; Movie S2). Furthermore, the addition of nanomolar concentrations of acetylcholine (ACh) or the selective ACh-receptor agonist carbachol resulted in muscle fiber contraction, demonstrating the functionality of the i-Myocytes (Movie S3). Similar results were obtained with i-Myocytes generated from MYOD1 OPTi-OX hiPSCs (Figure S3F). Importantly, induction efficiency did not decrease over extended culture periods (>50 passages, Figure 3H), thus demonstrating the robustness and reproducibility of this method. Finally, we noted that the levels of the MYOD1 transgene following induction positively correlated with conversion efficiency (Figures 3I and 3J), which highlights the importance of a robust gene-delivery method. In conclusion, these data demonstrated that the OPTi-OX platform enables robust and rapid forward programming of hPSCs into skeletal myocytes.

14Encouraged by our results deriving neurons and myocytes, we sought to utilize the same overexpression system to develop a forward programming protocol for oligodendrocytes. Oligodendrocytes are of critical importance for CNS function and their loss or dysfunction plays a key role in many neurological diseases. Unlike neurons, protocols for efficient generation of human oligodendrocytes from renewable sources remain an unmet need: currently available hPSC differentiation protocols are extremely long (up to 200 days) and yield heterogeneous cell populations.

15We generated OPTi-OX hPSCs bearing inducible SOX10 either alone or in combination with OLIG2 in the form of a polycistronic expression cassette (Figure 4A). Although cells induced with SOX10 alone robustly expressed the oligodendrocyte precursor (OPC) marker O4 after 10 days of induction, these cells failed to differentiate into myelin-expressing cells and died (Figure 4A). In contrast, the OLIG2-SOX10 overexpressing cells progressed from an O4-positive progenitor stage into a mature CNP/MBP-positive phenotype after 20 days of induction (Figure 4A). Moreover, gene expression analysis confirmed that OLIG2-SOX10 OPTi-OX hPSCs induced in oligodendrocyte medium supplemented with the mitogens PDGFaa and FGF2 first passed through an OPC-like stage, during which they remained proliferative and co-expressed typical OPC markers (Figures 4B–4F). Remarkably, following withdrawal of mitogens, i-OPCs differentiated into mature oligodendrocytes, expressing the typical myelin-associated proteins (Figures 4G–4M).

16OPTi-OX is the result of a systematic effort to optimize gene expression in hPSCs. It relies on a dual GSH-targeting strategy for the Tet-ON system, overcoming the limitations of viral-mediated transgene delivery forward programming protocols and allows stronger and more controlled transgene overexpression compared with previous targeting approaches. Table S2 compares the gene-delivery methods that have been used for transcription factor expression in different hPSC forward programming approaches. Moreover, site-specific insertion of the two components of the inducible gene expression system minimizes genomic off-target effects and together with the chemically defined medium compositions enhances the reproducibility of the protocols.

17The functionality of our platform is exemplified through the production of several cell types. First, we show that NGN2 and MYOD1 OPTi-OX hPSCs can be used as an inexhaustible source for highly scalable, rapid, single-step, virus-free, chemically defined, fully reproducible, and deterministic generation of i-Neurons and i-Myocytes.

18Finally, we successfully applied the OPTi-OX platform to develop a forward programming protocol for generating human oligodendrocytes. Recent studies demonstrated that forced expression of transcription factors allows direct conversion of rodent fibroblasts, and primary human fetal neural stem cells into OPCs, but the reprogramming of renewable human cell sources into oligodendrocytes has not been reported. While i-OPCs undergo the expected morphological changes, and express mature markers in monocultures in vitro, further characterization of the cells using co-culture models and transplantation into myelin-deficient mutants is needed.

19Human oligodendrocytes are of considerable interest for several applications. The efficiency and speed of the presented forward programming system will enable high-throughput drug screens and toxicology testing, in vitro modeling of hereditary leukodystrophies, and the development of cell-transplantation strategies.

20Transcription factor combinations for direct cellular reprogramming into many cell types of clinical interest are now available, including cardiomyocytes, pancreatic β cells, and hepatocytes. We anticipate that the OPTi-OX platform will be applicable for the generation of many other cell types. Overall, the presented method can provide the basis for inexhaustible, high-throughput, homogeneous, and large-scale manufacturing of many human cell types.

21Targeting of the hROSA26 and AAVS1 locus was performed as described recently. Targeting of the hROSA26 locus was done by nucleofection. Neomycin-resistant colonies were picked and screened by genotyping. Correctly hROSA26-rtTA-targeted clones were subsequently targeted with the inducible transgene cassette in the AAVS1 locus by lipofection. Resulting puromycin-resistant colonies were picked and re-analyzed by genotyping.

22Inducible overexpression was performed with dual GSH-targeted OPTi-OX hPSCs. Expression of inducible transgenes was prompted by adding dox to the culture medium. For forward programming into neurons, skeletal myocytes, and oligodendrocytes, standard medium conditions for the derivation of the respective cell types were used. Gene and protein expression analysis was performed as described recently. Please refer to the Supplemental Experimental Procedures for details on culture conditions and analysis techniques.

23M.P. conceived the study, designed and performed experiments, analyzed data, and wrote the first draft of the manuscript. D.O. designed and performed experiments and analyzed the data. A.B. designed and performed experiments, analyzed data, and wrote the manuscript. J.M.T. performed additional experiments. R.A.P. provided expert advice. L.V. supervised and supported the study. M.R.N.K. conceived, supervised, supported the study, and finalized the manuscript.