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Differentiation of human induced pluripotent stem cells into hypothalamic vasopressin neurons with minimal exogenous signals and partial conversion to the naive state

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.


Results
Primed FNDI-specific hiPSCs fail to aggregate and differentiate poorly using conventional methods. Differentiation of mESCs and hESCs into hypothalamic neurons was achieved by SFEBq 28,33 , but there were differences between the methods. With minimal exogenous signals, mESCs differentiated into hypothalamic neurons whereas hESCs failed to form aggregates. As previously reported, addition of KSR, BMP4 and SAG caused hESCs to differentiate into hypothalamic precursors, but these complicated steps of differentiation did not follow the embryonic development 28,33 . Therefore, we speculated that differentiation into AVP neurons should be based on the concept of minimizing the effects of exogenous signals. First, we used FNDI iPSCs (FDI-02) to investigate which differentiation protocols were appropriate because our aim in this study was to differentiate AVP neurons from FNDI hiPSCs and identify mutant proteins. We confirmed that FDI-02 differentiated into FOXG1-positive telencephalic progenitor cells with continuous addition of 5% KSR (Fig. S2), as reported for hESCs 33 . To minimize the effects of exogenous signals, we next tried to reduce KSR concentration (0.7%, 1.0%, 1.5%, 2.5%) or shorten its incubation period (day 0-3) (Fig. S2). When the concentration of KSR at day 0 was reduced (less than 2.5%) or KSR-free gfCDM was used after day 3, the aggregates collapsed or formed cysts (Fig. S2).
Our previous study using miPSCs showed that suspending miPSC colonies to approximately 10-20 cells, rather than isolating them to a single cell for SFEBq, can facilitate miPSC aggregation 29 . Accordingly, SFEBq using clumps was performed to prevent collapse and cyst formation. When the KSR concentration at day 0 was 2.5% and KSR-free gfCDM was used after day 3, the clumps quickly aggregated. As this aggregate grew, it partially collapsed but kept partially aggregated. Even under these conditions, FNDI-iPSCs (FDI-02) differentiated into FOXG1-positive telencephalic progenitors (Fig. S2). When a lower concentration of KSR (0.7%, 1.0%, 1.5%) was used from day 0, the aggregates collapsed or formed cysts. Although SFEBq using clumps could ease the effects of reduced KSR concentration to some extent, it was difficult for primed FNDI-iPSCs (FDI-02) to differentiate into hypothalamic neurons with minimal exogenous signals.
Partial conversion from primed to naive state improves the differentiation of FDI-02 into hypothalamic precursors. To minimize the exogenous signals needed for FNDI-iPSCs (FDI-02) differentiation, we assessed a method that enables human pluripotent stem cells to be treated in the same manner as mouse pluripotent stem cells.
Compared with conventional mESCs, human pluripotent stem cells show some distinctive features, including differences in developmental identity. In particular, mEpiSC derivation 30,31 shows alternative pluripotency, which is associated with primitive streak-stage late epiblasts 34 . The terms 'naive' and 'primed, ' which correspond to mESCs and mEpiSCs, respectively, refer to the early and late stages of epiblast ontogeny 32 . Human pluripotent stem cells share defining features with primed mEpiSCs rather than with naive mESCs. We hypothesized that these methods could help hiPSCs differentiate into hypothalamic neurons by minimizing exogenous signals.
In this study, we employed RSeT™ feeder-free medium (ST-05975; STEMCELL Technologies, Vancouver, Canada), a kit for primed to naive conversion based on the articles by Chan et al., Gafni [35][36][37][38] . Primed FNDI-iPSCs (FDI-02) cells were cultured under feeder-free conditions using mTeSR1™ culture medium. Thereafter, the medium was replaced with RSeT™. The colony morphology changed from flat to tightly packed and domed. We conducted immunohistochemistry on passage(P)4 and 9. TFCP2L1 and KLF4, naive markers 39 , were expressed ( Fig. 1A-F). We also confirmed the significant increase of naive markers expression (TFCP2L1 and STELLA) by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (Fig. 1G,H) on P3, 4, and 5. These results demonstrated multiple features of the naive state. However, these features are less obvious than in previous reports [35][36][37][38] and the KLF4 expression was not localized in the nucleus. Therefore, the conversion by this kit might be partial. SFEBq was performed with partially naive FNDI-iPS (FDI-02) single cells and primed FDI-02 clumps. The use of a medium containing neither KSR nor Y-27632, a Rho-associated kinase (ROCK) inhibitor, from an early stage was compared with a medium containing both throughout the culture ( Fig. 2A). Even when the initial concentration of KSR was reduced to 0.7%, the single cells and clumps quickly aggregated under all conditions. (B,C) On day 30, naive aggregates showed significantly fewer changes, such as cyst formation or collapse (n = 3; three 96-well plates, that is, 288 aggregates are analyzed). Mean ± SEM. *P < 0.05; **P < 0.01, two-tailed unpaired t-test. On day 30, primed aggregates scarcely expressed BRN2; however, BRN2 expression was observed by conversion to the naive state (D), and was significantly increased by shortening the period of KSR and Y-27632 treatment (n = 8) (E). Mean ± SEM. *P < 0.05, **P < 0.01, two-tailed unpaired t-test. OTP (green), BRN2 (red), FOXG1 (white). The increased expression of the AVP precursor marker, BRN2, indicated that minimization of exogenous signals improved the efficiency of differentiation into hypothalamic-like cells. For all relevant panels, nuclear counterstaining was performed with DAPI (blue). Scale bars, 100 µm. www.nature.com/scientificreports/ Most primed aggregates formed cysts or collapsed over time, whereas partially naive aggregates maintained a healthy state. The partially naive aggregates showed significantly fewer changes on day 30 ( Fig. 2B,C). Aggregates were evaluated by immunohistochemistry. The expression of BRN2, an AVP precursor marker 40,41 , was significantly increased in partially naive aggregates in the shortened period condition with KSR and Y-27632 ( Fig. 2D,E). We demonstrated that not only the conversion by the naive conversion kit but also the minimization of exogenous signals was essential for the differentiation into AVP neurons. We also confirmed that the partially naive aggregates contained very few FOXG1 positive cells. This is consistent with increased hypothalamic differentiation with minimal exogenous signals.
In summary, conversion by the naive conversion kit enabled minimization of exogenous signals in SFEBq with hiPSCs. Furthermore, the minimization of exogenous signals improved the efficiency of differentiation into hypothalamic-like cells.
Hypothalamic organoids derived from FDI-02 expressed mutant NPII. We have previously reported that the addition of FGF8b to late-stage culture improves the efficiency of AVP neuron differentiation 29 . Therefore, we adopted this method and established an overall protocol (Fig. 3A).
Human iPSC cell line, 201B7 was converted as FDI-02 and SFEBq was performed according to the established protocol as a control. Dorsal hypothalamic progenitor (PAX6+, NKX2.1−) and AVP precursor (OTP+, BRN2+) cells were sequentially identified (Fig. 3B,C). On day 150, we observed cells co-expressing AVP, NPII, and copeptin, which are components of pro-AVP, and thus achieved differentiation into AVP neurons (Fig. 3D). Using the same method, FDI-02 differentiated into AVP neurons. Simultaneously, we confirmed the expression of other hypothalamic markers such as CRF, NPY, AgRP, TRH, and Orexin ( Fig. 3E-I), which demonstrated successful differentiation to AVP neurons as well as hypothalamic organoids. In FNDI iPSCs, we also confirmed the sequential differentiation into hypothalamic organoids (Fig. S3). Finally, we proved the expression of mutant NPII, which was not expressed in 201B7-derived AVP neurons (Fig. 4).

Discussion
In this study, we investigated a novel perspective in the differentiation of AVP neurons, regarding whether cells were in a naive or primed state before differentiation, rather than considering the early or later stages of the differentiation protocol 28,29,33 . We found two advantages. First, the trial of the conversion of cells from primed to naive state solved problems such as decreased survival, collapse of aggregates, and cyst formation by minimizing exogenous signals in the SFEBq method for the differentiation of human iPSCs into AVP neurons. Second, even after solving these problems, the strict removal of exogenous signals further increased the rate of cells positive for BRN2, which is an essential transcription factor for AVP differentiation [40][41][42][43][44] . These findings indicate an improved efficiency of AVP neuron differentiation.
The differentiation potential of pluripotent stem cells varies among cell lines 45 . When differentiating the rostral part of the central nervous system such as the hypothalamus, the use of naive cells could be one way to solve this problem. It is difficult to differentiate the primed hESCs/hiPSCs into rostral hypothalamic neurons because they cannot survive in culture conditions with minimal exogenous signals. Historically, it is known that dissociation of primed hESCs causes apoptosis due to the Rho-high/Rac-low state 46 . Thus, treatment with ROCK inhibitors is very effective for the dissociation process of primed hESCs 47 . Using a ROCK inhibitor, primed hESCs/hiPSCs can be differentiated into non-rostral nervous systems such as the telencephalon 17,18 , cerebellum 22 , and spinal cord 24 . However, for inducing the rostral part of the central nervous system, such as AVP neurons, additional conditions are required to induce the rostral edge of the neural plate 28,29 . In the primed hESC/hiPSC cultures, use of the ROCK inhibitor alone cannot maintain their aggregation in the state of minimal exogenous signals. Nutrients such as KSR must be added to some extent for their survival, resulting in their differentiation into FOXG1-positive cerebral tissues. Additional BMP4 and SHH signals are necessary to primed hESCs for repositioning from the cerebrum to the hypothalamus 33 . This means that a two-step process takes place in which the hESCs, once posteriorised, are repositioned anteriorly (Fig. S1B). In this report, the conversion of hiPSCs by the naive conversion kit, which minimized the addition of ROCK inhibitors and KSRs, may have resulted in a simpler and more straightforward direction of differentiation and improved the efficiency of differentiation to the rostral hypothalamus.
The stability of the naive conversion is a significant issue. The kit manufacturer recommends monitoring their karyotype after 10 passages. Therefore, we used iPSCs within 10 passages after conversion and confirmed their expression of naive markers at P9 by immunohistochemistry. The converted hiPSCs in this study kept their feature when we conducted SFEBq.
FNDI is a hereditary neurodegenerative intractable disease for which there is currently no fundamental treatment. Analyses of our FNDI model mice, which possess the Cys98stop mutation 2,3 , revealed that mutant NPII was accumulated in the ER-associated compartment (ERAC) in AVP neurons 4,5 . In this study, we clearly demonstrated mutant NPII expression in FNDI-disease specific hiPSC-derived AVP neurons. Accumulation of mutant proteins in the ER is implicated in the pathophysiology of many diseases, including FNDI 1,6-13,48,49 . Furthermore, novel ER stress-reducing drugs have been reported 50 . Therefore, FNDI-disease specific hiPSC-derived AVP neurons are a promising human model of ER stress and are a valuable resource for drug development. We plan to analyze characteristic structures, such as the ERAC, which was confirmed in mice by electron microscopy, in FNDI-specific hiPSC-derived AVP neurons. We would also like to undertake a functional investigation for pathological analysis and drug development in future studies.
There is a limitation about the naive state in this study. The conversion was performed with RSeT™ feeder-free medium [35][36][37][38] . Although we were able to identify multiple features of the naive state, the conversion to the naive state by this kit might be partial as we described in Results. Even this partial conversion was enough to play an Further studies are needed to determine which way of naive conversion for hiPSC is the best in differentiating human rostral hypothalamus. In addition, the positive areas of hypothalamic markers, including AVP, were localized to a small part of the aggregates. Based on this study, we will continue to improve the differentiation method, analyze the expression pattern of mutant proteins in detail, and research drug reactivity.

Methods
These experiments were approved by the ethics committee of Nagoya University and performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all subjects.  based on ethical approval from Nagoya University Committee (2013-0228-2). To establish patient-derived iPSCs, peripheral blood monocular cells of the patient were reprogrammed using episomal vectors (expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28, EBNA1, and p53shRNA) as described previously 57,58 . The iPSCs showed embryonic stem cell-like morphology and normal karyotype (Fig. S4A). These cells expressed the undifferentiated markers (Fig. S4B,C) and could differentiate into the three germ layers in vitro (Fig. S4D), indicating that these cells were pluripotent (deposited in RIKEN BRC as HPS1011 and HPS1904). In this study, we used a cell line named FDI-02, which corresponds to HPS1904. As a control, we used the 201B7 cell line  www.nature.com/scientificreports/ dish (354277; Corning, Corning, New York, USA), and incubated under 5% CO 2 at 37 °C. After 24-36 h, the mTeSR1 medium was replaced with RSeT™ feeder-free medium (ST-05975; STEMCELL Technologies) and cells were incubated under hypoxic conditions (5% O 2 , 5% CO 2 ) at 37 °C. The medium was changed every second day. Reset to the naive state was confirmed by changes in colony morphology and immunohistochemistry. For passaging, naive hiPSCs were harvested using TrypLE Express (12605-010; Thermo Fisher, Waltham, Massachusetts, USA). Cells were counted and 1.1 × 10 6 cells were distributed on a 10 cm Matrigel-coated dish. The medium was changed after 24-36 h and thereafter, every second day. This passaging procedure was performed every 4-6 days. These cells were used within 10 passages for the differentiation experiments.
Immunohistochemistry. Aggregates were fixed with 4% PFA for 10-15 min and embedded in OCT compound (4583; Sakura Finetek, Tokyo, Japan). Ten micron thick sections were cut using a cryostat, mounted on slides, and fixed in 4% PFA for 10 min.
Primary antibodies were used against the following molecules (with dilutions): AVP (T5048; guinea pig;