Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

doi:10.21203/rs.3.rs-2085626/v1

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Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

https://doi.org/10.21203/rs.3.rs-2085626/v1

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Background: Degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) in Parkinson's disease (PD) is responsible for motor and cognitive impairment. Replacing the dopaminergic cell population in the SNpc to restore normal dopamine levels is a potential therapeutic approach. However, improving neuronal integration still requires a reliable cell source for transplantation and a profound understanding of the effects of the local microenvironment on transplanted cells. We have previously shown that embryoid bodies (EBs)-derived cells from mouse embryonic stem cells overexpressing the dopaminergic transcription factor Lmx1a engrafted into SNpc develop tyrosine hydroxylase (TH)-positive phenotype. In the present work, we transplanted EBs-derived cells from genetically engineered human embryonic stem cells (hESCs), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO). We determined their potential to differentiate into TH-expressing neurons in the SNpc of an in vivo PD model.

Methods: EBs-derived cells from genetically the engineered hESCs-LFO cell line were transplanted, and their neuronal differentiation potential was determined in the SNpc of an in vivo PD model with 6-hydroxy dopamine (6-OHDA). Three rat groups were designed as follows: Untreated (healthy rats), sham (rats administered with saline solution), and 6-OHDA (rats lesioned with 6-OHDA). A one-way ANOVA test was performed for statistical analysis.

Results: Neural rosettes, a fundamental developmental hallmark of neuroepithelial tissue, were found at 7 and 15 days post-transplantation (dpt) in ~ 70% of the transplanted brains in all three conditions: Untreated, sham, and 6-OHDA. The majority of the neural rosettes corresponded to the lumen formation stage. In comparison, no graft survival was observed in EB transplants derived from unmodified hESCs. Interestingly, at 30 dpt, hESC-LFO engrafted cells showed neuronal morphology and positive immunolabeling for TH in all the brains exhibiting surviving transplants: 10% 6-OHDA rats, 0% sham, and 100% untreated rats.

Conclusions: Overall, our results show that overexpression of LFO factors favors short-term survival while strongly initiating neural differentiation of hESC-derived cells in SNpc surviving grafts by forming neural rosettes and differentiating into TH-positive cells.

Neural differentiation

human embryonic stem cells

stem cell transplantation

neural rosettes

transcription factors.

Parkinson's disease (PD) is a neurodegenerative disorder affecting the mesencephalic dopaminergic neurons in the substantia nigra pars compacta (SNpc), resulting in motor (bradykinesia, tremor, and difficulty initiating movement [1, 2]) and non-motor symptoms (abnormalities, behavioral changes, sleep disturbances) [3]. New strategies to slow disease progression or even replace lost cells are urgently needed, as current pharmacological treatments are only symptomatic and have long-term side effects [4, 5].

Among the promising therapies are cell-based regenerative approaches, including the engraftment of differentiated dopaminergic neurons into the striatum [6, 7] and the SNpc [8], the latter being particularly interesting as it is the necessary niche to achieve physiological regeneration of mesencephalic dopaminergic neurons. This requires the generation of axonal projections to the striatum, which as has been partially demonstrated previously [9, 10].

Human embryonic stem cells (hESCs) represent a reliable and reproducible cell source [11], as recent work has evaluated their potential for cell replacement in the SNpc at various stages of neuronal development [12, 13]. However, little is known about the viability and differentiation potential of hESCs at earlier stages when transplanted into the SNpc, where its integration into the nigrostriatal pathway improved.

In this respect, embryoid bodies (EBs), cell aggregates derived from hESCs, provide a well-studied model of embryonic development in vitro, expressing early embryonic markers in a compartmentalized manner [14, 15]. EBs derived from ESCs constitute an essential initial step in vitro directed dopaminergic differentiation protocols [16–18]. In this line of work, we have shown that EBs-derived cells from mouse ESCs (mESCs) survive and differentiate into neurons in the adult rodent SNpc in vivo, but they lack the differentiation potential to acquire a dopaminergic cell fate [19]. Hence, other strategies may be required to drive dopaminergic differentiation of EBs-derived cells in the SNpc.

The regulatory transcription factors LIM Homeobox Transcription Factor 1 Alpha (LMX1A), Forkhead Box A2 (FOXA2), and Orthodenticle Homeobox 2 (OTX2) (collectively referred to as LFO) are known to be essential for controlling the specification and maintaining the dopaminergic mesencephalic phenotype [20, 21]. On this subject, we recently evaluated the dopaminergic differentiation potential of transplanted EBs-derived cells from mESCs overexpressing murine LMX1A [18]. We found that LMX1A promotes dopaminergic differentiation in untreated SNpc in vivo. Furthermore, Zhu et al. demonstrated that simultaneous overexpression of human transcription factors LMX1A, FOXA2, and OTX2 enhanced dopaminergic differentiation in vitro of EBs-derived cells from a hESC cell line (named hESC-LFO) [20]. The TALEN-engineered transgene cassette containing the transcription factors was under the transcriptional control of the nestin promoter to ensure its transcription during early neural differentiation in vitro [20].

In the present work, we tested to what extent overexpression of LFO transcription factors promoted tyrosine hydroxylase (TH)-positive differentiation (a sign of early dopamine synthesis) of EBs derived from the hESC-LFO cell line after engraftment into the SNpc of untreated, sham, and 6-hydroxydopamine (6-OHDA)-injured rats (the latter since the 6-OHDA lesion favors the differentiation potential) [18]. Interestingly, we found that these cells differentiated into neural rosettes, an essential step in neuroepithelial cell development, at 7 and 15 days post-transplantation (dpt). Moreover, at 30 dpt, the transplanted cells exhibited neuronal morphology, and TH-expressing cells were observed in both microenvironmental conditions (lesioned and untreated) in all the brains in which the transplant survived. In contrast, hESCs without transcription factors did not survive. Thus, our results indicate that overexpression of midbrain LFO transcription factors enhances survival and promotes robust neural differentiation by forming neural rosettes that progress to differentiate into TH-expressing cells.

Embryoid body culture

For cell transplantation, two hESC lines (hESC and hESC-LFO) developed by Zhu et al. [20] were used; similarly, the protocol standardized by this group was performed for EB generation. Briefly, hESCs were cultured on mouse embryonic feeders for 7 days. Afterward, cell colonies were manually dissected and passaged onto feeder-free Geltrex-coated plates with STEMFLEX medium (Thermo Fisher). Colonies were then cultured for 4 days. To form EBs, 1 mg/mL of collagenase IV (Sigma-Aldrich) dissolved in E6 medium (Thermo Fisher) was added to the culture plate to rinse the hESCs. The detached colonies were transferred to ultra-low adhesion culture wells (Sigma-Aldrich) and cultured for 4 days with E6 medium supplemented with 15% inactivated fetal bovine serum (Thermo Fisher). On the fourth day, EBs were rinsed, disaggregated, and resuspended in 5 µl of phosphate-buffered saline (PBS, Thermo Fisher) for transplantation [22].

6-hydroxydopamine lesion in SNpc of rats and human embryonic stem cell transplantation

Adult male Wistar rats (250 g) were housed under standard conditions: 12:12 light/dark cycle, controlled room temperature (27°C), and ad libitum access to food and water. Animals were chemically lesioned with 40 µg of 6-OHDA (Sigma-Aldrich) mixed with 32 µg/µL of L-ascorbate (JT Baker) in a total volume of 0.5 µL of saline solution, this group was designated as "6-OHDA". Two control groups were included for hESC grafts. In the "sham" group, 0.9% saline solution was used instead of 6-OHDA, while in the "untreated" group only cells were administrated into the SNpc. Before surgical procedures, the animals were anesthetized with xylazine (8 mg/kg, PiSA, Mexico City, Mexico) and ketamine (100 mg/kg, PiSA). 6-OHDA administration and cell transplantation into the rat brains were performed by stereotaxic surgery; SNpc coordinates were 4.8 rostral, 1.6 lateral to bregma, 8.1 ventral [23]. Each 6-OHDA infusion was injected at a rate of 0.125 mL/min into the left SNpc [18]. For cell transplantation, 3 µL of EB-derived cell suspension containing approximately 60,000 cells were injected using a 5 µL-Hamilton syringe (8929C51, Thomas Scientific). To prevent graft rejection, transplanted rats were subjected to daily oral cyclosporine A (50 mg/mL, PiSA) treatment (100 mg/kg/day) until brain removal. All procedures were conducted in accordance with the National Institute of Health Guide for the Use and Care of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Institute of Cell Physiology (permit number: MGC65-19) of the National Autonomous University of Mexico. Animal numbers were kept to a minimum and all efforts were made to reduce animal suffering.

Rotational behavior

Two weeks after 6-OHDA administration, an amphetamine-induced rotational test was performed to confirm dopaminergic depletion in the nigrostriatal pathway. To measure dopaminergic imbalance, animals were injected intraperitoneally with 4 mg/kg amphetamine (Sigma-Aldrich). The number of turns was recorded for 90 min using a custom-made computerized image and motion recognition system [24]. Animals were evaluated two weeks after injury, and only those with more than 400 ipsilateral turns were selected for transplantation procedures.

Histology and immunofluorescence staining

To analyze grafted EBs-derived cells at different developmental stages, rats were euthanized at 7, 15, or 30 dpt with pentobarbital and perfused intracardially with 100 mL of 0.1 M PBS followed by 100 mL of 4% paraformaldehyde (PFA, Millipore). Brains were postfixed in 4% PFA and cryoprotected by sequential 24-h incubations in 10%, 20%, and 30% sucrose (in PBS). 40 µm slices were taken from the coronal plane of brains and collected in an antifreeze solution. Standard hematoxylin and eosin (H&E, Abcam) staining procedures were performed to visualize the cell morphology of the transplanted tissues. Human-specific antibody STEM121 (Takara Bio) was used to distinguish between engrafted human and endogenous rat cells.

Fontana-Masson staining kit (Sigma-Aldrich) was used to detect melanin aggregates following the manufacturer's instructions [25]. Briefly, slides were washed with distilled water, placed in 10% Fontana silver nitrate at 56 ºC for two h, and rinsed three times in distilled water. The slides were then incubated in a 1% gold chloride solution for 1 min and rinsed in distilled water. They were then placed in a 5% sodium thiosulfate solution for 1 min and thoroughly rinsed in distilled water twice. Finally, staining was performed with eosin solution, and the sections were washed with distilled water and subsequently dehydrated and mounted.

Immunofluorescence assays were performed as previously described [24]. Brain slices were rinsed three times in PBS/0.3% Triton X-100 (Sigma-Aldrich), treated with antigen retriever in citrate buffer (BioSB) at 65°C for 30 min, and washed three times with PBS. The tissues were then incubated overnight at four °C with primary antibodies diluted in 0.1 M PBS blocking solution with 0.2% donkey serum (Gibco). Antibodies used in this experiment are listed in Supplementary Table 1. After rinsing three times in PBS, the slides were incubated for two h at room temperature with the corresponding Alexa fluor secondary antibody: 458 anti-mouse (Millipore) or 645 anti-goat (Millipore). Finally, the sections were rinsed with PBS, and four ′,6-diamidino-2-phenylindole (DAPI; 1:10,000; Biostatus Limited) was added to the final rinse for counterstaining cell nuclei. The tissue was mounted onto Superfrost slides (Thermo Fisher) with Black Diamond fluorescent mounting medium (Abcam).

Image acquisition and cell counting

Images for counting individual cells were captured on an Olympus FV1000 (Upright BX61WI and inverted IX81) confocal microscope. For tissue transplants and neural rosettes visualization, a Zeiss Observer Z.1 spinning disk microscope and a Keyence BZ-X700 microscope were used. ImageJ/Fiji software was used to quantify and classify neural rosettes and for 3D reconstructions. The inset images (Fig. 5C) were taken from a stack of 29 slides with a width of 204.8 µm. The central area shown corresponds to the first slide, and the lower region to slide 29. The 3D model of the transplant (Fig. 5C) was made using a stack of 94 images. A one-way ANOVA test was performed for statistical analysis.

hESC-LFO cells show neural rosette formation at 7 and 15 days post-transplantation

In order to determine whether LFO factors could induce TH-positive differentiation of EBs in vivo, we transplanted EBs-derived cells from the hESC or hESC-LFO lines into the SNpc of untreated, saline-injected (sham), or 6-OHDA injured rats (Fig. 1A, B, F, G). At 7 or 15 dpt, immunofluorescence for STEM121 (not shown) and H&E staining showed that there was no graft survival in the brains of animals transplanted with the hESC-derived EBs [see Supplementary Fig. 1 and Supplementary Table 2, n = 43].

In contrast, grafts derived from hESC-LFO EBs survived and evolved into neural rosettes in untreated, sham, and 6-OHDA lesioned rats at 7 and 15 dpt. The STEM121 signal indicating the human origin of the graft was present in all neural rosettes (Fig. 1C, D). Furthermore, at 7 and 15 dpt, ~ 70% of the transplanted brains in all three conditions, untreated (10 rats), sham (16 rats), and 6-OHDA lesioned rats (16 rats), exhibited grafts containing neural rosettes in the SNpc (see Supplementary Table 2).

The marker Zonula occludens 1 (ZO-1) is expressed in the lumen of neural rosettes generated in vitro. Therefore, we used it as a bona fide marker for neural rosettes [26–30]. Indeed, ZO-1 has consistently expressed in the lumen of neural rosettes in 6-OHDA lesioned rats at 7 and 15 dpt (Fig. 1E), indicating that EBs-derived cells from hESC-LFO were able to differentiate into neural rosettes in the SNpc of adult rats. 87.5% (n = 8) of 6-OHDA rats presented neural rosettes at 7 dpt, and 75% (n = 8) at 15 dpt. Similarly, 75% (n = 8) of sham rats presented neural rosettes at 7 dpt, and 87.5% (n = 8) at 15 dpt (Fig. 1F) [see Supplementary Table 2]. No significant differences were found between the groups at the two time points. Although there were no significant differences in the number of neural rosettes between groups (p = 0.466), there was a trend indicating that the 6-OHDA condition had more neural rosettes per graft than the sham or untreated conditions (Fig. 1G).

Neural rosettes express dopaminergic lineage markers

Immunofluorescent assays were carried out to determine if LMX1A and OTX2 transcription factors were expressed in the neural rosette formation. Indeed, LMX1A (Fig. 2) and OTX2 (Fig. 3) were consistently observed in the nuclei of neural rosettes at 7 dpt (Fig. 2, 3A-L) and 15 dpt (Fig. 2, 3M-X) in 60% untreated (n = 10), 81.2% sham (n = 16), and 81.2% 6-OHDA (n = 16) rats. We observed that LMX1A and OTX2 expression colocalized with DAPI signals only in STEM121-positive cells. In contrast, cells without STEM121 signal (endogenous rat tissue) did not express these transcription factors, indicating that LMX1A and OTX2 were only expressed in grafted cells (Fig. 2, 3). However, it was not possible to determinate if the expression of LMX1A and OTX2 derives from the endogenous locus or the introduced transgene.

To test whether neural rosettes mimicked neuronal differentiation through the neuroblast stage, we assessed the expression of doublecortin (DCX), a protein involved in neural migration expressed in neuroblasts and young neurons [19]. Indeed, DCX was found to colocalize with STEM121 at 7 dpt (Fig. 4A-L) and 15 dpt (Fig. 4M-X) in untreated (Fig. 4A-D, M-P), sham (Fig. 4E-H, Q-T), and 6-OHDA (Fig. 4I-L, U-X) animals. Interestingly, the signal was especially prominent adjacent to the lumen of neural rosettes at 7 dpt (Fig. 4B, F, J). In contrast, at 15 dpt, DCX expression was predominantly observed surrounding the rosettes (Fig. 4N, R, V). A remarkable rearrangement of these structures is apparent, as cells seem to migrate from the lumen towards the outer zone, while at 30 dpt, the graft no longer showed a rosette formation. However, instead, cells appeared spindle-shaped and with projections, suggesting that LFO contributes to the progressive development of neural rosettes in immature neurons [31].

Neural rosettes are mainly at the differentiation stage of lumen formation

To analyze the stage of differentiation of neural rosettes, we categorized and quantified the neural rosettes found in untreated, sham, and 6-OHDA-lesioned rats at 7 and 15 dpt, according to the classification of neural rosettes proposed by Hribková et al, which constitutes of 5 stages of differentiation found in vitro: cell intercalation, cell constriction, cell polarization, cell elongation, and lumen formation [30]. We classified the morphology of neural rosettes based on the cytoplasmatic signal given by the STEM121 antibody and the nuclear DAPI signal. We observed that the neural rosettes present in our transplants were only at the stages of cell polarization, cell elongation, and lumen formation (Fig. 5A). Additionally, we observed that a certain amount of neural rosettes were morphologically different from the proposed classification and named them “other types” (Fig. 5A).

We found that the mean number of neural rosettes was significantly higher (p = 0.044) at the lumen formation stage than at other stages in sham and 6-OHDA-lesioned rats at 7 and 15 dpt (Fig. 5B). Moreover, at 7 dpt, 6-OHDA-lesioned rats showed a significantly higher number of neural rosettes (p = 0.031) at the lumen formation stage (15.25 ± 3.16) than in sham (7.85 ± 1.71) or untreated rats (5.9 ± 2.48) (Fig. 5B). These results indicates that neural rosettes formed by EBs derived from hESC-LFO mainly differentiated to the lumen-forming stage at 7 and 15 dpt.

Next, we analyzed 3D stacks of neural rosettes (n = 5) to assess differences in morphology given by the slide level obtained during 2D micrographs acquisition. We confirmed that the majority of the neural rosettes correspond to the lumen formation stage, as the center of the neural rosettes shows a lumen surrounded by cells with elonged morphology (Fig. 5C). Taking into account our observations, we show a graphic representation of the differentiation of the engrafted cells into neural rosettes (Fig. 5D).

hESC-LFO grafts express tyrosine hydroxylase and neuromelanin at 30 days post-transplantation

At 30 dpt, the graft survived only in a few animals, in 1 of 11 (n = 11) 6-OHDA-lesioned rats and 2 of 2 (n = 2) untreated rats (Fig. 6), whereas sham rats showed no graft survival (n = 5, data not shown). Next, we assessed the expression of LMX1A, OTX2, DCX, and TH, the limiting enzyme of dopamine synthesis. Interestingly, at this stage, cells maintained some expression of LMX1A (Fig. 6A), OTX2 (Fig. 6B), and robust expression of DCX (Fig. 6C, D-G). Notably, all the brains exhibiting surviving transplants showed neuronal morphology and positive immunolabeling for TH, although not uniformly throughout the graft, as not all cells were labeled (Fig. 6H-K). The morphology of the engrafted cells visualized in a 3D reconstruction (Fig. 6H, J) shows TH- and STEM121-labeled neuronal projections colocalizing in the graft, suggesting that hESC-LFO cells were differentiating into TH-positive neurons. It is important to highlight that quantification of TH-positive cells was not possible due to the high density of cell bodies and their projections (Fig. 6H-K) and the TH-stippled pattern exhibited by cells in the untreated SNpc, which does not allow adequate delineation of the projections (Fig. 6H, I). However, it is possible to observe in cells transplanted from the lesioned SNpc a significant amount of cells in which TH-STEM121 signals were colocalizing (Fig. 6J, K), in contrast to cells transplanted in the untreated SNpc, where only a few cells were observed (Fig. 6H, I).

Furthermore, we observed that brain samples exhibiting cell survival and TH-positive cells also had a dark pigmentation resembling neuromelanin (Fig. 6A, B), a bioproduct of dopamine oxidation in SNpc neurons [32]. A Fontana-Masson neuromelanin staining [33, 34] confirmed its presence (Fig. 6L, M). Therefore, at 30 dpt, grafted cells survived and differentiated into neuronal-like cells with emerging projections, expressing TH and neuromelanin.

In this study, we have shown that overexpression of LFO transcription factors enabled the survival and neuronal specification of EBs-derived cells from the hESC-LFO line engrafted into SNpc, highlighting that the expression of LMX1A, FOXA2, and OTX2 transcription factors support differentiation toward TH-expressing cells in vivo. The hESC-LFO-derived EBs generated neural rosettes, a well-known developmental structure of human neural precursor cells in vitro [28, 29, 35]; they also expressed DCX, indicating neuroblast and immature neuron stages; and matured into neuron-like cells that expressed TH and produced neuromelanin [36].

The expression of the LFO transcription factors, individually and together, is necessary for the specification and maintenance of the dopaminergic mesencephalic phenotype in vitro [20] and in vivo, as they regulate several genes with a critical role in the mesencephalic dopaminergic lineage, such as Pitx3, Girk2, or Nurr1 [21]. Here, LFO factors were necessary to initiate neural development and TH-positive neuronal differentiation and, remarkably, for graft survival. Indeed, Domansky et al. [37] identified that FOXA2 is crucial for survival at the specification and maintenance stages. Similarly, it has been observed that LMX1A regulates the survival of adult midbrain dopaminergic neurons [38], whereas OTX2 inactivation results in midbrain dopaminergic cell death [39]. Our results may indicate that overexpression of LFO factors together generated a synergistic effect, favoring the survival of hESC-LFO-derived EBs. However, further investigation will be required to evaluate the effect generated by the overexpression of individual transcription factors.

The formation of neural rosettes in our grafts indicated the development of neuroepithelial tissue, with a trend for a higher prevalence of neural rosettes in the injured SNpc than in untreated SNpc. To our knowledge, this is the first report of differentiation via neural rosette formation in SNpc after transplantation of EBs derived from hESCs. As mentioned above, Hribková et al. [30] classified neural rosettes according to their shape and timing of neuronal development and proposed five stages of neural rosette formation [30]. In our experiments, neural rosettes in a single graft showed several of the described stages of differentiation, such as polarization, elongation, and lumen formation. Overall, we found that the lumen formation stage was significantly more prevalent at 7 and 15 dpt.

Furthermore, our 3D analysis confirmed that the neural rosettes were composed of a 3D structure with the lumen zone located at the center of the neural rosette, similar to the neural rosettes obtained with in vitro organoid experiments by Knight et al. [40]. It is essential to highlight that undifferentiated ESC grafts can generate noncancerous overgrowths that may be detrimental to brain function [41]. Nonetheless, we did not observe tumorigenic growths at the different times of EB transplantation analyzed. On the contrary, the morphology development from neural rosettes towards individual cells, and the positive labeling to DCX and TH with leading processes, suggest that the cells are in a progressive process of differentiation.

Indeed, our group has previously reported that untreated SNpc promotes neuronal differentiation of EBs derived from mESCs up to 30 dpt [19]. Interestingly, we recently observed that the SNpc of 6-OHDA-lesioned rats is a permissive niche for initiating dopaminergic differentiation of EBs derived from mESCs overexpressing LMX1A at six dpt [18]. Here, we observed a significant increase in the number of neural rosettes formed in the lesioned SNpc, indicating the development of the lumen-forming stage of neural rosettes in the 6-OHDA-lesioned group was favored as compared to the controls. This indicates that the lesioned SNpc may have relevant factors for the differentiation of engrafted cells, as observed in previous work [18, 41]. Therefore, further research is required to identify the correlation between the 6-OHDA lesion and the microenvironmental cues to facilitate cell differentiation in the SNpc.

In addition to LMX1A, OTX2, and TH expression, at 30 dpt, the neuromelanin found in the grafted area was a direct indication of dopamine oxidation and, consequently, an indirect suggestion of dopamine synthesis by the grafted cells, which is an indispensable feature for dopaminergic neurons, as has also been seen in organoids directed towards a dopaminergic phenotype [42, 43]. However, given that the transplant survived in only 16% (n = 18) of the rats at 30 dpt, there could be apoptotic signaling being triggered due to intrinsic characteristics of the cells (e.g., increased susceptibility to cell death as occurs in the dopaminergic phenotype), which could explain why at 7 and 15 dpt there was survival and differentiation as neural rosettes. In contrast, at 30 dpt the survival rate decreased considerably. Similarly, this difference between 7 and 30 dpt could mean that more potent immunosuppression is needed to overcome possible immune reactions that promote transplant cell death, as the rats used in this study were pharmacologically immunosuppressed [44]. Early immunosuppression, or a combination of cyclosporine A use with other immunosuppressors, could favor cell survival to improve it at longer time intervals [45].

This study provides evidence that LFO transcription factors promote survival and support TH-positive differentiation potential of hESCs in the SNpc through the formation of neural rosettes. However, further evaluation of overexpression-driven differentiation in vivo, including the impact of microenvironmental cues and long-term engraftment assessment to determine functional cell integration, is required to understand how these transcription factors drive survival and neuronal specification in vivo.

6-OHDA, 6-hydroxydopamine; DCX, doublecortin; dpt, days post-transplantation; EB, embryoid body; FOXA2, Forkhead Box A2; hESCs, human embryonic stem cells; H&E, hematoxylin and eosin; hESC-LFO, human embryonic stem cells overexpressing transcription factors LMX1A, FOXA2, and OTX2; LMX1A, LIM Homebox Transcription Factor; mESCs, mouse embryonic stem cells; OTX2, Orthodenticle Homebox; PD, Parkinson’s disease; PFA, paraformaldehyde; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; ZO-1, Zonula occludens.

Ethics approval and consent to participate

All studies were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Animal Care and Use Committee of the Institute of Cell Physiology (Permit number: MGC65-19) of the National Autonomous University of Mexico. Animal numbers were kept to a minimum and all efforts were made to reduce animal suffering. All methods are reported in concurrence with ARRIVE guidelines for describing animal experiments [46].

Consent for publication

No applicable.

Availability of data and materials

All data generated and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This project was funded by DGAPA-PAPIIT, grant numbers IN207116 and IN211419, and by CONACyT, grant number A1-S-10064. RRA (CVU 768446), CAMT (CVU 699541), LDBC (CVU 767791), and FJPG (CVU 1037918) were supported by grants from the National Council of Science and Technology.

Authors’ contributions

MGC conceptualized and supervised the research study. RRA, CAMT, and FZ performed the research. RRA, CAMT, LDBC, BS and MGC analyzed the data. RRA, CAMT, FJPG, BS, and MGC wrote the manuscript. MPR provided technical assistance through the experiments. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors are grateful to Claudia Rivera for providing the animals. The authors thank Dr. Andrés Saraleguí and Dr. Arturo Pimentel of the Laboratorio Nacional de Microscopía Avanzada, and Dr. Abraham Rosas Arellano at the Unidad de Microscopía (Instituto de Fisiología Celular, UNAM) for their technical assistance in confocal imaging. The authors also thank to Sandra Daniela Rodríguez Montaño, Omar Collazo-Navarrete, Gilda Guerrero-Flores, Francisco Pérez-Eugenio, and Ana María Escalante for their technical assistance throughout this work.

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No competing interests reported.

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Human embryonic stem cells overexpressing dopaminergic transcription factors survive and differentiate in the substantia nigra in vivo

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Abstract

Figures

Background

Materials And Methods

Embryoid body culture

6-hydroxydopamine lesion in SNpc of rats and human embryonic stem cell transplantation

Rotational behavior

Histology and immunofluorescence staining

Image acquisition and cell counting

Results

hESC-LFO cells show neural rosette formation at 7 and 15 days post-transplantation

Neural rosettes express dopaminergic lineage markers

Neural rosettes are mainly at the differentiation stage of lumen formation

hESC-LFO grafts express tyrosine hydroxylase and neuromelanin at 30 days post-transplantation

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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