Embryoid Body Cells from Human Embryonic Stem Cells Overexpressing Dopaminergic Transcription Factors Survive and Initiate Neurogenesis via Neural Rosettes in the Substantia Nigra.

Transplantation of immature dopaminergic neurons or neural precursors derived from embryonic stem cells (ESCs) into the substantia nigra pars compacta (SNpc) is a potential therapeutic approach for functional restitution of the nigrostriatal pathway in Parkinson's disease (PD). However, further studies are needed to understand the effects of the local microenvironment on the transplanted cells to improve survival and specific differentiation in situ. We have previously reported that the adult SNpc sustains a neurogenic microenvironment. Non-neuralized embryoid body cells (EBCs) from mouse ESCs (mESCs) overexpressing the dopaminergic transcription factor Lmx1a gave rise to many tyrosine hydroxylase (Th+) cells in the intact and damaged adult SNpc, although only for a short-term period. Here, we extended our study by transplanting EBCs from genetically engineered naive human ESC (hESC), overexpressing the dopaminergic transcription factors LMX1A, FOXA2, and OTX2 (hESC-LFO), in the SNpc. Unexpectedly, no graft survival was observed in wild-type hESC EBCs transplants, whereas hESC-LFO EBCs showed viability in the SNpc. Interestingly, neural rosettes, a developmental hallmark of neuroepithelial tissue, emerged at 7- and 15-days post-transplantation (dpt) from the hESC-LFO EBCs. Neural rosettes expressed specification dopaminergic markers (Lmx1a, Otx2), which gave rise to several Th+ cells at 30 dpt. Our results suggest that the SNpc enables the robust initiation of neural differentiation of transplanted human EBCs prompted to differentiate toward the midbrain dopaminergic phenotype.

Embryoid Body Formation from Mouse and Human Pluripotent Stem Cells for Transplantation to Study Brain Microenvironment and Cellular Differentiation.

Human embryonic stem cell (hESC) and human-induced pluripotent stem cell (hiPSC) technologies have a critical role in regenerative strategies for personalized medicine. Both share the ability to differentiate into almost any cell type of the human body. The study of their properties and clinical applications requires the development of robust and reproducible cell culture paradigms that direct cell differentiation toward a specific phenotype in vitro and in vivo. Our group evaluated the potential of mouse ESCs (mESCs), hESCs, and hiPSCs (collectively named pluripotent stem cells, PSCs) to analyze brain microenvironments through the use of embryoid body (EB)-derived cells from these cell sources. EB are cell aggregates in 3D culture conditions that recapitulate embryonic development. Our approach focuses on studying the midbrain dopaminergic phenotype and transplanting EB into the substantia nigra pars compacta (SNpc) in a Parkinson's disease rodent model. Here, we describe cell culture protocols for EB generation from PSCs that show significant in vivo differentiation toward dopaminergic neurons.

Transcriptomic analysis reveals new hippocampal gene networks induced by prolactin.

Prolactin (Prl) is a pleiotropic hormone with multiple functions in several tissues and organs, including the brain. In the hippocampus, Prl has been implicated in several functions, including neuroprotection against excitotoxicity in lactating rats and in Prl-treated ovariectomized animals. However, the molecular mechanisms involved in Prl actions in the hippocampus have not been completely elucidated. The aim of this study was to analyse the hippocampal transcriptome of female Prl-treated ovariectomized rats. Transcriptomic analysis by RNASeq revealed 162 differentially expressed genes throughout 24 h of Prl treatment. Gene Ontology analysis of those genes showed that 37.65% were involved in brain processes that are regulated by the hippocampus, such as learning, memory and behaviour, as well as new processes that we did not foresee, such as glial differentiation, axogenesis, synaptic transmission, postsynaptic potential, and neuronal and glial migration. Immunodetection analysis demonstrated that Prl significantly modified microglial morphology, reduced the expression of Cd11b/c protein, and altered the content and location of the neuronal proteins Tau, Map2 and Syp, which are involved in axogenic and synaptic functions. This novel delineation of Prl activity in the hippocampus highlights its importance as a neuroactive hormone, opens a new avenue for understanding its actions and supports its participation in neuronal plasticity of this brain area.

The Substantia Nigra Is Permissive and Gains Inductive Signals When Lesioned for Dopaminergic Differentiation of Embryonic Stem Cells.

Transplantation of dopaminergic (DA) cells into the striatum can rescue from dopamine deficiency in a Parkinson's disease condition, but this is not a suitable procedure for regaining the full control of motor activity. The minimal condition toward recovering the nigrostriatal pathway is the proper innervation of transplanted DA neurons or their precursors from the substancia nigra pars compacta (SNpc) to their target areas. However, functional integration of transplanted cells would require first that the host SNpc is suitable for their survival and/or differentiation. We recently reported that the intact adult SNpc holds a strong neurogenic environment, but primed embryonic stem cells (ie, embryoid body cells, EBCs) could not derive into DA neurons. In this study, we transplanted into the intact or lesioned SNpc, EBCs derived from embryonic stem cells that were prompt to differentiate into DA neurons by the forced expression of Lmx1a in neural precursor cells (R1B5/NesE-Lmx1a). We observed that, 6 days posttransplantation (dpt), R1B5 or R1B5/NesE-Lmx1a EBCs gave rise to Nes+ and Dcx+ cells within the host SNpc, but a large number of Th+ cells derived only from EBCs exogenously expressing Lmx1a. In contrast, when transplantation was carried out into the 6-hydroxidopamine-lesioned SNpc, the emergence of Th+ cells from EBCs was independent of exogenous Lmx1a expression, although these cells were not found by 15 dpt. These results suggest that the adult SNpc is not only a permissive niche for initiation of DA differentiation of non-neuralized cells but also releases factors upon damage that promote the acquisition of DA characteristics by transplanted EBCs.

Functional determination of the differentiation potential of ventral mesencephalic neural precursor cells during dopaminergic neurogenesis.

The ventral mesencephalic neural precursor cells (vmNPCs) that give rise to dopaminergic (DA) neurons have been identified by the expression of distinct genes (e.g., Lmx1a, Foxa2, Msx1/2). However, the commitment of these NPCs to the mesencephalic DA neuronal fate has not been functionally determined. Evaluation of the plasticity of vmNPCs suggests that their commitment occurs after E10.5. Here we show that E9.5 vmNPCs implanted in an ectopic area of E10.5 mesencephalic explants, retained their specification marker Lmx1a and efficiently differentiated into neurons but did not express the gene encoding tyrosine hydroxylase (Th), the limiting enzyme for dopamine synthesis. A proportion of E10.5-E11.5 implanted vmNPCs behaved as committed, deriving into Th+ neurons in ectopic sites. Interestingly, implanted cells from E12.5 embryos were unable to give rise to a significant number of Th+ neurons. Concomitantly, differentiation assays in culture and in mesencephalic explants treated with Fgf2+LIF detected vmNPCs with astrogenic potential since E11.5. Despite this, a full suspension of E12.5 vmNPCs give rise to DA neurons in a similar proportion as those of E10.5 when they were transplanted into adult brain, but astrocytes were only detected with the former population. These data suggest that the subventricular postmitotic progenitors present in E12.5 ventral mesencephalon are unable to implant in embryonic explants and are the source of DA neurons in the transplanted adult brain. Based on our findings we propose that during DA differentiation committed vmNPCs emerge at E10.5 and they exhaust their neurogenic capacity with the rise of NPCs with astrogenic potential.

Mouse embryonic stem cell-derived cells reveal niches that support neuronal differentiation in the adult rat brain.

A neurogenic niche can be identified by the proliferation and differentiation of its naturally residing neural stem cells. However, it remains unclear whether "silent" neurogenic niches or regions suitable for neural differentiation, other than the areas of active neurogenesis, exist in the adult brain. Embryoid body (EB) cells derived from embryonic stem cells (ESCs) are endowed with a high potential to respond to specification and neuralization signals of the embryo. Hence, to identify microenvironments in the postnatal and adult rat brain with the capacity to support neuronal differentiation, we transplanted dissociated EB cells to conventional neurogenic and non-neurogenic regions. Our results show a neuronal differentiation pattern of EB cells that was dependent on the host region. Efficient neuronal differentiation of EB cells occurred within an adjacent region to the rostral migratory stream. EB cell differentiation was initially patchy and progressed toward an even distribution along the graft by 15-21 days post-transplantation, giving rise mostly to GABAergic neurons. EB cells in the striatum displayed a lower level of neuronal differentiation and derived into a significant number of astrocytes. Remarkably, when EB cells were transplanted to the striatum of adult rats after a local ischemic stroke, increased number of neuroblasts and neurons were observed. Unexpectedly, we determined that the adult substantia nigra pars compacta, considered a non-neurogenic area, harbors a robust neurogenic environment. Therefore, neurally uncommitted cells derived from ESCs can detect regions that support neuronal differentiation within the adult brain, a fundamental step for the development of stem cell-based replacement therapies.

[Osteogenesis of human vascular endothelial cells in culture]. / Osteogénesis en cultivo de endotelio vascular humano.

INTRODUCTION: Mesenchymal stem cells have the potential to differentiate into several types of cells including osteoblasts. These stem cells have cell surface markers found on cells of endothelial and subendothelial origin of the umbilical cord vein. Taking this into consideration we have postulated that human umbilical vein endothelial cells (HUVEC) could present osteogenic differentiation as well. Gene activation that could drive osteogenic differentiation is regulated by exogenous and endogenous factors. OBJECTIVE: The induction osteogenesis in HUVEC. MATERIAL AND METHODS: We used: a) an osteogenic medium containing 0.1 microM dexamethasone, 10 microM beta-glycerophosphate, 50 microM L-ascorbic-acid 2-phosphate, 20% MCGS serum; and b) a treatment with DNA demethylating agents hydralazine and 5'-aza-2'-deoxycytidine (0.39-200 microM). Phenotypic characteristics of HUVEC were their spindle and stellate shapes with fine homogenous cytoplasm, typically associated with fibroblast-like cells. RESULTS: The control cells (without osteogenic treatment) exhibited little extracellular matrix, whereas the osteogenically treated cells appeared shortened and flattened, and they were surrounded by extracellular matrix that subsequently became mineralized in vitro. After 28 days in culture, morphologic and histochemical studies confirmed that osteogenic medium had a strong stimulatory effect on the alkaline phosphatase activity of endothelial cells, a very early marker of cell differentiation into the osteogenic lineage. Hydralazine and 5'-aza-2'-deoxycytidine, two drugs utilized in chromatin remodeling leading to gene re-expression of inactivated DNA hypermethylated islands, did not favor osteoblast differentiation. CONCLUSION: Our study shows that HUVEC can differentiate along an osteogenic lineage and thus provide an alternative source for cell-based therapies and tissue engineering strategies.