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Stem Cell Reports
Stem Cell Reports
Stem Cell Reports
2213-6711
Elsevier

S2213-6711(24)00115-2
10.1016/j.stemcr.2024.04.010
Report
Forced LMX1A expression induces dorsal neural fates and disrupts patterning of human embryonic stem cells into ventral midbrain dopaminergic neurons
Rifes Pedro 15
Kajtez Janko 15
Christiansen Josefine Rågård 1
Schörling Alrik 124
Rathore Gaurav Singh 1
Wolf Daniel A. 2
Heuer Andreas 23
Kirkeby Agnete agnete.kirkeby@sund.ku.dk
124∗
1 Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW) and Department of Neuroscience, University of Copenhagen, 2200 Copenhagen N, Denmark
2 Department of Experimental Medical Science, Lund University, 22184 Lund, Sweden
3 Behavioural Neuroscience Laboratory, Department of Experimental Medical Sciences, Lund University, 22184 Lund, Sweden
4 Wallenberg Center for Molecular Medicine, Department of Experimental Medical Sciences, Lund University, 22184 Lund, Sweden
∗ Corresponding author agnete.kirkeby@sund.ku.dk
5 These authors contributed equally

16 5 2024
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© 2024 The Authors
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https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

The differentiation of human pluripotent stem cells into ventral mesencephalic dopaminergic (DA) fate is relevant for the treatment of Parkinson’s disease. Shortcuts to obtaining DA cells through direct reprogramming often include forced expression of the transcription factor LMX1A. Although reprogramming with LMX1A can generate tyrosine hydroxylase (TH)-positive cells, their regional identity remains elusive. Using an in vitro model of early human neural tube patterning, we report that forced LMX1A expression induced a ventral-to-dorsal fate shift along the entire neuroaxis with the emergence of roof plate fates despite the presence of ventralizing molecules. The LMX1A-expressing progenitors gave rise to grafts containing roof plate-derived choroid plexus cysts as well as ectopically induced TH-positive neurons of a forebrain identity. Early activation of LMX1A prior to floor plate specification was necessary for the dorsalizing effect. Our work suggests using caution in employing LMX1A for the induction of DA fate, as this factor may generate roof plate rather than midbrain fates.

Associated podcast

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Highlights

• Forced LMX1A expression during neural iPSC differentiation induced dorsal fate

• Ventral-to-dorsal fate shift was observed along the entire rostro-caudal axis

• Dorsalizing effect resulted from early outcompetition of the ventral FOXA2 program

• Forced LMX1A prevented the formation of DA neurons from midbrain progenitors

• Forced LMX1A expression resulted in choroid plexus-like cysts in transplants

Want to make a dopamine neuron through forced reprogramming? Then think twice before employing the transcription factor LMX1A for this purpose. In this article, Kirkeby and colleagues show that forced expression of LMX1A in hPSCs can abrogate midbrain dopamine neuron patterning and causes fate switch into roof plate neural fates and choroid plexus.

Keywords

dopamine
Parkinson’s disease
reprogramming
neural tube ventral patterning
human embryonic stem cells
induced neurons
transplantation
cell replacement therapy
Published: May 16, 2024
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pmcIntroduction

The ventral midbrain (vMB) dopaminergic (DA) neuron lineage has attracted much attention in the last decades, due to its association with Parkinson’s disease, which is hallmarked by neurodegeneration of the DA midbrain nucleus termed substantia nigra. Extensive molecular characterization of the progenitor population located in the ventral floor plate of the developing neural tube has led to the discovery of numerous genes implicated in the patterning, specification, and maturation of DA neurons (Blaess and Ang, 2015). Out of a multitude of transcription factors expressed locally and involved in the regionalization of the vMB domain, the LIM-homeodomain LMX1A has been identified as a determinant factor for the specification of the DA progenitors and neurons (Andersson et al., 2006; Doucet-Beaupre et al., 2015). Although the Lmx1a mouse mutant Dreher displays a mild reduction of the vMB DA neuronal population, compensated by the paralog Lmx1b (Yan et al., 2011), ectopic expression of Lmx1a can drive the formation DA neurons in the ventral neural tube, but not in dorsal neural tube, of mouse and chicken embryos (Andersson et al., 2006; Lin et al., 2009; Nakatani et al., 2010). Moreover, forced expression of Lmx1a in mouse embryonic stem cells (mESCs) (Andersson et al., 2006; Friling et al., 2009) and mouse fetal fibroblasts, together with other neuronal reprogramming factors (Caiazzo et al., 2011; Dell’Anno et al., 2014; Kim et al., 2011), can in vitro induce the formation of cells expressing tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Altogether, these observations have supported several reprogramming studies using LMX1A to induce DA progenitors or neurons from human cells in vitro (Caiazzo et al., 2011; Friling et al., 2009; Pfisterer et al., 2011; Rivetti di Val Cervo et al., 2017). However, attempts to apply LMX1A and other factors for obtaining DA neurons in vivo—through either direct in vivo reprogramming or transplantation of induced neurons (iNs)—have generated disappointing results with only very few TH+ neurons obtained (Pereira et al., 2014; Torper et al., 2013). Thus, the regional neural identity resulting from forced expression of LMX1A in human cells remains elusive.

In this study, we applied a human embryonic stem cell (hESC)-based in vitro model of early human neural patterning (Rifes et al., 2020) to investigate the outcome of forced LMX1A expression along the entire rostro-caudal neuraxis. We found that despite the presence of ventralizing factors, forced LMX1A expression caused a global ventral-to-dorsal fate shift of neural progenitors, regardless of their rostro-caudal regionality. We further confirmed the dorsalizing effect of LMX1A through transplantation studies and found that LMX1A induced the formation of choroid plexus cysts as well as ectopic TH expression in forebrain cells, while abrogating the formation of DA neurons from midbrain cells.

Results

Lentiviral expression of LMX1A in an in vitro model of early human neural patterning

We have demonstrated previously that neural differentiation of a layer of hESCs under a microfluidic-controlled gradient of the small-molecule WNT activator CHIR99021 (0–2 μM) recapitulates the earliest steps of rostro-caudal neural tube patterning in vitro and generates a coherent neural tissue ranging from forebrain to hindbrain (Rifes et al., 2020). In this model, termed MiSTR (microfluidic stem cell regionalization), the global addition of sonic hedgehog (SHH) agonists (SHH and purmorphamine) results in an hESC-derived neural tissue with a ventral identity, generating a rostro-caudal neuraxis at the level of the floor plate (Rifes et al., 2020). To investigate the patterning effect of forced LMX1A expression along the entire ventral human neuroaxis, we delivered a lentiviral construct of LMX1A (LV-LMX1A) to hESCs and subjected these cells to MiSTR differentiation (Figure 1A). The LV-LMX1A construct applied contained a target sequence for the pluripotency-specific microRNA mir292 within the 3′-UTR. This construct has previously been shown to efficiently silence transgenic protein translation in pluripotent cells and to activate expression only 3–5 days after initiation of differentiation (Sachdeva et al., 2010). Indeed, while expression of the LV-LMX1A mRNA was detected by quantitative reverse-transcription PCR (RT-qPCR) in both hESCs and differentiated day-14 cells (Figure 1B), we confirmed that LMX1A was detected at protein level only in differentiated cells but not in hESCs (Figure 1C). Furthermore, we demonstrated that the human phosphoglycerate kinase (hPGK) promoter induced LV-LMX1A expression at physiological levels comparable to endogenous LMX1A levels in vMB cells (Figure 1B). Surprisingly, we found through OTX2 immunolabelling and RT-qPCR analysis that ventralized MiSTR tissue originating from hESCs expressing LV-LMX1A did not induce a global midbrain signature but instead maintained the overall rostro-caudal patterning from forebrain to hindbrain (Figures 1D and 1E), similar to what has previously been demonstrated for wild-type hESCs (Rifes et al., 2020). However, when assessing the dorsoventral identity of the tissues, it was revealed that LV-LMX1A expression resulted in a strongly reduced expression of ventral markers such as NKX2-1, SIX6, FOXA2, and SHH (Figures 1E and 1F). Concomitantly, there was an induction of dorsal neural tube and roof plate markers (e.g., GDF7 and WNT3A), which were absent in the ventralized control tissue without LV-LMX1A (Figure 1F). Our MiSTR results indicate that, despite the presence of ventralizing molecules, forced expression of LMX1A did not induce global vMB fates but in contrast abrogated ventralization and provoked a ventral-to-dorsal fate shift along the entire rostro-caudal neuraxis in human neural tissue.Figure 1 Forced lentiviral expression of LMX1A in a stem cell-based in vitro model of ventral neural tube patterning induces dorsal identity

(A) A lentiviral construct with LMX1A under control of 4x mir292 target sequences was applied to hESCs (referred to as LV-LMX1A). The LV-LMX1A-expressing hESCs were seeded into the MiSTR model of human rostro-caudal patterning of the neural tube. After 14 days, the tissue was cut into 5 regional portions along the rostro-caudal gradient for RT-qPCR analysis, or a longitudinal strip was taken for immunofluorescence.

(B) Cycle threshold values (Ct) from RT-qPCR analysis using primers specific for endogenous LMX1A (LMX1A-UTR) or for lentiviral LMX1A (LMX1A-WPRE) expression. Results are displayed for hESCs and MiSTR day-14 samples (section C) with and without LV-LMX1A expression. hESC data are from at least 3 independent experiments while MiSTR data are from at least 7 independent experiments.

(C) Immunolabeling for LMX1A displays immunoreactive expression only in LV-LMX1A cells at day 5 of differentiation indicating the protein is expressed only once cells exit pluripotency. No expression was detected in hESCs nor differentiated control cells.

(D) Normalized expression of forebrain, midbrain, and hindbrain markers (RT-qPCR) along the A-E regions of ventralized LV-LMX1A MiSTR tissue at day 14. n = 7 tissues from independent experiments.

(E) Whole-mount MiSTR tissue immunolabelling for the forebrain/midbrain marker OTX2 and ventral marker NKX2-1 in control and LV-LMX1A tissues. Note that Matrigel-derived autofluorescence is noticeable as a diffuse red haze on the OTX2-negative side.

(F) Normalized RT-qPCR expression values show induction of dorsal markers in LV-LMX1A MiSTR tissues compared to control tissue. n = 10 (control tissues), n = 7 (LV-LMX1A) from independent experiments.

Grafts from LV-LMX1A-expressing cells confirm a ventral-to-dorsal fate shift

To validate whether the observed ventral-to-dorsal fate shift observed in the MISTR model also held true upon in vivo transplantation, we patterned individual cultures of hESCs (control and LV-LMX1A cells) toward either ventral forebrain (vFB) and vMB or ventral hindbrain (vHB) fates (Figure 2A; Figure S1), using a previously published protocol (Kirkeby et al., 2012). These cells were then transplanted into the striatum of adult rats which had been unilaterally lesioned with 6-hydroxydopamine (6-OHDA) to eliminate endogenous DA neurons, thereby enabling better visualization of graft-derived DA neurons (Figure 2B). The transplanted cells from all 6 groups survived implantation into the rat brain and gave rise to human neuron-containing grafts after 6 weeks (Figure 2C). Interestingly, we observed the formation of large cystic structures in grafts of the LV-LMX1A group (5 out of 9 animals), which were not present in the control grafts (0 out of 44 animals, Figure 2D). These cysts (4 out of 5, Figure S2A) were lined with human non-neuronal cells (HuNu+/hNCAM−) expressing the choroid plexus markers transthyretin (TTR) and OTX2, indicating that the cysts represented ectopic hESC-derived ventricular structures (Figures 2E and 2F). The cysts were also positive for other mature choroid plexus markers such as tight junction protein Zonula occludens 1 (ZO-1), Na–K–Cl cotransporter (NKCC1), and Na/K ATPase α1 (ATP1A1) (Figure S2B). Given that choroid plexus cells are derived from dorsal roof plate structures in the developing embryo, these findings substantiated the ventral-to-dorsal fate shift observed in the LV-LMX1A MiSTR tissue (Figure 1F). The LV-LMX1A-induced generation of choroid plexus cells particularly in progenitors of hindbrain fate further explained the presence of OTX2+ cell patches in the hindbrain region of the LV-LMX1A MiSTR tissue (Figure 1E).Figure 2 Ventrally differentiated LV-LMX1A hESCs form cystic structures with choroid plexus marker TTR upon grafting into rat brain

(A) Control hESCs and LV-LMX1A hESCs were patterned toward different ventral fates (forebrain, midbrain, and hindbrain; vFB, vMB, vHB, respectively) and prepared as cell suspensions for transplantation to the rat brain.

(B) Cell suspensions of patterned cells were injected into the striatum of 6-hydroxydopamine-lesioned rat brains at 4 weeks post lesion, and the grafts were analyzed 6 weeks post-transplantation.

(C) Survival and neural identity of the grafts were verified for all 6 conditions of in vitro-differentiated cells through immunolabeling for human neural cell adhesion molecule (hNCAM). CL, cyst lumen.

(D) Immunolabeling for a human-specific epitope (human nuclei, HuNu) in 6-week-old grafts from control and LV-LMX1A cells revealed the presence of cystic structures only in LV-LMX1A-derived grafts. The number of grafts containing cysts in each group was quantified relative to total number of animals with grafts in each group.

(E) HuNu+ LV-LMX1A cells lining the cysts in vMB grafts displayed immunoreactivity for the choroid plexus marker TTR.

(F) LV-LMX1A cells lining the human-specific NCAM-stained grafts strongly expressed the choroid plexus marker OTX2.

(G) Immunohistochemistry reveals robust presence of TH+ cells in control vMB-patterned grafts and to lesser extent in LV-LMX1A vFB-patterned grafts. The graph on the right displays the quantification of the total number of TH+ cells in control and LV-LMX1A grafts. Data are presented as mean ± SEM, n = 3 grafted animals.

(H) Immunolabeling for the enzymes TH and AADC, which are essential for dopamine synthesis, reveals an absence of AADC in the LV-LMX1A-derived vFB TH+ cells. In contrast, AADC is present in TH+ cells of grafts derived from control hESCs differentiated toward vMB.

We next analyzed the grafts for the presence of dopamine neurons. In line with our previous studies (Grealish et al., 2014; Kirkeby et al., 2012), when using wild-type control hESCs, we observed TH+ neurons only in grafts derived from vMB-patterned progenitors, but not in vFB or vHB grafts (Figure 2G). In contrast, vMB-patterned grafts from LV-LMX1A-expressing cells were surprisingly devoid of TH+ neurons, indicating again a loss of ventral fates induced by LMX1A. Interestingly, LV-LMX1A expression in the vFB setting induced the formation of TH+ neurons (Figure 2G) that were, in contrast to transplanted hESC-derived vMB DA neurons, negative for the dopamine synthesis enzyme aromatic l-amino acid decarboxylase, AADC (Figure 2H). This indicated that the ectopically induced forebrain-derived TH+ neurons were not of a bona fide vMB-dopamine-producing phenotype. In conclusion, the in vivo data thus further corroborated our in vitro MiSTR observations and demonstrated that forced expression of LMX1A not only failed to induce vMB fates but instead initiated a dorsal transcriptional program in the cells in vitro and in vivo.

Forced LMX1A expression overrides early ventralizing transcriptional program

Taking advantage of the in vitro MiSTR model to access events prior to the observed ventral-to-dorsal fate shift induced by LV-LMX1A expression, we reassessed the dynamics of the expression of ventral and dorsal markers at earlier time points in the system (day 2 and day 6 of differentiation; Figure 3A). RT-qPCR analyses revealed that a ventral transcriptional program with induction of NKX2-1, NKX6-1, SIX6, FOXA1, and FOXA2 was initiated in both wild-type and LV-LMX1A-expressing MiSTR tissue at day 6 of differentiation. However, by day 14 of differentiation, these markers were almost completely lost in the LV-LMX1A tissues and instead replaced by the induction of dorsal roof plate markers such as GDF7, SOX10, and WNT1/3A (Figure 3A). Interestingly, the early expression of floor plate genes (FOXA1/2, SHH) by day 6 tended to be reduced, albeit not statistically significant, in the LV-LMX1A tissues (Figure 3B), indicating that forced LMX1A expression abrogated the positive feedback loop for ventralization between day 6 and 14. We further explored the effect of timing of forced LMX1A expression by applying an inducible Tet-O-Lmx1a system (Figure S3A). Through addition of doxycycline, we induced Lmx1a expression at day 3, 6, or 9 of differentiation and analyzed the cells at day 14 (Figure 3C). Immunocytochemistry for NKX2.1, FOXA2, and PAX6 showed that early induction of Lmx1a expression led to dorsalization (day 3 and to lesser extent day 6) while activation of Tet-O-Lmx1a on day 9 had only a minor effect (Figure 3D). Analysis of the cells at day 5 showed that induction of Lmx1a expression caused significant activation of the WNT pathway (i.e., increase in WNT1 and LEF1) as well as antagonism of the SHH pathway (i.e., inhibition of PTCH1) already at two days after initiation of doxycycline treatment (Figure 3E). In line with this, supraphysiological activation of SHH signaling through combination of high SHH and purmorphamine was able to initiate earlier and stronger expression of FOXA2 and thereby prevented the dorsalizing effect from forced expression of Lmx1a starting on day 3 (Figure S3B). These data indicate that ventral and dorsal transcriptional programs are in competition and that forced LMX1A expression during early stages can prematurely activate strong WNT signaling, thereby overriding an emerging ventral floor plate program and inducing a ventral-to-dorsal fate shift.Figure 3 Temporal expression of ventral and dorsal markers in ventralized LV-LMX1A MiSTR tissue

(A) Normalized RT-qPCR expression values from day 2, 6, and 14 of forebrain, midbrain, and hindbrain markers along the A-E regions of ventralized MiSTR tissue displays suppression of ventral markers in LV-LMX1A tissues between days 6 and 14. n = 4 (day 2), 5 (day 6), 7 (day 14) LV-LMX1A MiSTR tissues from independent experiments.

(B) Non-normalized fold-change values underlying the heatmap in (A) show the transient induction of ventral markers NKX2-1, FOXA1/2, and SHH in ventral MiSTR differentiation, comparing control (+SHH) and LV-LMX1A MiSTR tissues, at 6 and 14 days. Control, non-ventralized MiSTR tissue (no SHH, black) is shown only for visual comparison. Data represented as mean ± SEM. Two-way ANOVA followed by post hoc Sidak test between +SHH control and LV-LMX1A. p-summary of comparison at each region (A-E) is shown above. n = 4 (day 2), 6 (day 6), 10 (day 14) control (+SHH) MiSTR tissues from independent experiments.

(C) Graphical representation of the experimental setup where Lmx1a expression was induced in differentiating Tet-O-Lmx1a hESCs by the addition of doxycycline from day 3, 6, or 9. Patterned cells were analyzed at day 14 and compared to control cells not treated with doxycycline.

(D) Immunolabeling of differentiated Tet-O-Lmx1a cells treated with doxycycline from day 3, 6, or 9, and an untreated control for FOXA2, NKX2.1, and PAX6 at day 14.

(E) Expression of genes (RT-qPCR) in the SHH signaling pathway (SHH, PTCH1, FOXA2) and WNT signaling pathway (WNT1, LEF1, SP8, PAX6) at day 5 in vMB-patterned Tet-O-Lmx1a cells with or without doxycycline from day 3. Data represented as mean ± SEM. Left y-axis (blue) applies to the blue columns and the right y-axis (red) refers to the red columns. n = 3 independent experiments.

Discussion

In this study, we provide evidence that early forced LMX1A expression at physiological levels in differentiating hESCs can result in the generation of a non-vMB DA progenitor fate, despite the presence of ventralizing factors in the medium and a lentiviral design that avoids precocious delivery of LMX1A in the pluripotent stage. Our MiSTR data demonstrated that forced LMX1A expression resulted in a clear ventral-to-dorsal shift along the entire embryonic neural axis, even though the tissue displayed an earlier induction of the ventral floor plate markers FOXA1/2 and SHH. Thereby, the transgenic LMX1A was capable of overriding the initiated floor plate program in early differentiating hESCs, while simultaneously inducing another, dorsal embryonic program. The competition between dorsal and ventral programs appeared to take place in a time window between days 3–6 of differentiation, where we also observed a strong WNT-activating effect of forced LMX1A expression. WNT1 is a direct transcriptional target of LMX1A (Chung et al., 2009), and we speculate that this premature activation of strong WNT signaling induced by LMX1A could be responsible for overriding the early ventral FOXA2/SHH program.

In mouse and chick, expression of Lmx1a in early neural tube development has been shown to initiate in the dorsal midline, the roof plate anlage, preceding the onset of expression in the vMB (Chizhikov and Millen, 2004; Doucet-Beaupre et al., 2015; Failli et al., 2002). In line with this, the spontaneous Lmx1a mouse mutant Dreher displays a notorious absence of roof plate structures (Millonig et al., 2000), while displaying only a moderate loss of vMB TH+ DA neurons, because of a partial compensatory effect of Lmx1b (Ono et al., 2007), which is expressed exclusively in the floor plate and also involved in midbrain DA neurogenesis (Smidt et al., 2000). Furthermore, forced LMX1A expression in embryonic neural tubes resulted in ectopic roof plate formation (Chizhikov and Millen, 2004), in line with our reported appearance of ectopic, LMX1A-induced cysts lined with choroid plexus-like cells in the vMB-patterned grafts. Moreover, lentiviral expression of Lmx1a in mESC-derived neural progenitors revealed a positive regulatory feedback loop with Wnt1 (Chung et al., 2009), which we also observed in our MiSTR tissue. However, Wnt1 is also expressed early in the roof plate (Parr et al., 1993), and, thus, the authors report that only co-delivery of Lmx1a, Foxa2, and Otx2 results in TH/AADC double-positive DA neurons (Chung et al., 2009). Therefore, the ventral-to-dorsal shift observed in our data may reflect the execution of the embryonic LMX1A program as a dorsal roof plate inducer, probably before the establishment of a full floor plate identity.

In summary, our data suggest caution in the use of LMX1A as a factor for DA reprogramming, given its capability to elicit dorsal identity as well as to generate misleading ectopic TH expression in forebrain-patterned neurons. In fact, the induction of ectopic TH expression in non-DA neurons seems to be a more general problem, as both mESCs and hESCs undergoing direct reprogramming with Ascl1, a non-cell-type-specific proneural transcription factor, yield a sizable fraction of TH+ iNs (Ng et al., 2021).

Throughout embryonic development, transcription factors are essential determinants for various tissues and cell types, depending on temporal, spatial, and molecular context. In our study, we demonstrate that the use of LMX1A for direct reprogramming entails a risk of activating multiple different embryonic programs, some of which may yield unintended outcomes. This emphasizes the importance of thoroughly analyzing the regionalizing consequences of reprogramming factors when applying them to generate subtype-specific cells in vitro and in vivo.

Experimental procedures

Resources availability

Lead contact

Further information and requests for resources and reagents should be directed to the lead contact, Agnete Kirkeby (agnete.kirkeby@sund.ku.dk).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate new datasets.

hESCs

H9 hESCs were maintained on Matrigel (Corning)-coated culture dishes in StemMACS iPS-Brew XF medium (Miltenyi Biotec) and passaged with EDTA (0.5 mM) once weekly. LV-LMX1A hESCs were generated through transduction of H9 hESCs with a lentiviral construct containing the human open reading frame (ORF) for LMX1A (Pfisterer et al., 2011), which yields forced LMX1A expression upon exit from pluripotency (Sachdeva et al., 2010).

Statistical analysis

All RT-qPCR data (fold over H9 ESCs) were managed in Excel and statistically analyzed using GraphPad Prism 9 software. Differences were considered significant for p < 0.05 (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). For analysis between A-E regions within one condition and time point, one-way ANOVA was performed on the original or LOG-transformed data, followed by Tukey’s multiple comparison test between all regions. Each dataset was tested for its normal distribution (Shapiro-Wilk test) and equal variances (Brown-Forsythe test), and LOG transformed if needed, before ANOVA. When normal distribution or equal variance was not achieved, the non-parametric Kruskal-Wallis analysis was performed, followed by a Dunn’s multiple comparison test. For analysis across MiSTR tissue regions and conditions, two-way ANOVA was performed on LOG-transformed data, followed by a post hoc Sidak’s multiple comparison test at each A-E region between conditions. All error bars represent SEM.

Other experimental procedures

LV-LMX1A and control H9 cells were differentiated into an in vitro model of human neural development, termed MiSTR (Rifes et al., 2020), or as adherent cultures toward vFB, vMB, and vHB progenitors and grafted into the striatum of a rat model of Parkinson’s disease (Kirkeby et al., 2012), as described previously. Other procedures and further details are described in the supplemental experimental procedures.

Supplemental information

Document S1. Supplemental experimental procedures and Figures S1–S3

Document S2. Article plus supplemental information

Acknowledgments

This study was supported by the 10.13039/501100009708 Novo Nordisk Foundation (NNF17CC0027852 , NNF21CC0073729 , and NNF18OC0030286 ), 10.13039/100012774 Innovation Fund Denmark (BrainStem: 4108-00008A ), the Strong Research Environment at Lund University MultiPark, the 10.13039/501100004359 Swedish Research Council (70862601/Bagadilico ), The Crafoord Foundation, the Segerfalk Foundation, the Tore Nilsson Foundation, the Sven-Olof Janson Foundation, the 10.13039/501100003554 Lundbeck Foundation (R350-2020-963 and R380-2021-1267 ), and the 10.13039/501100000780 European Union (H2020 , NSC-Reconstruct GA no. 874758 ). A.H. was supported by a post-doctoral stipend from the 10.13039/501100003748 Swedish Society for Medical Research and a Starting grant by the 10.13039/501100004359 Swedish Research Council (VR2016-01789 ). We would like to thank Nanna MacAulay and Trine Lisberg Toft for NKCC1 and Na/K ATPase antibodies and Novo Nordisk for the LMX1A antibody. We thank Brenda Finlay for producing choroid plexus organoids and Malin Parmar for support with facilities and intellectual input to the study.

Author contributions

P.R. and A.K. designed the study. P.R., J.K., J.R.C., A.S., G.S.R., A.H., D.A.W., and A.K. performed experiments. P.R., J.K., and A.K. wrote the manuscript.

Declaration of interests

A.K. is the owner of Kirkeby Cell Therapy APS, performs paid consultancy to Novo Nordisk A/S, and is a co-inventor on patents WO2016162747A2/A3 and WO2019016113A1 on the generation of DA cells for treatment of PD.

Supplemental information can be found online at https://doi.org/10.1016/j.stemcr.2024.04.010.
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