Alexandre Hiroaki Kihara

Human Adult Stem Cells from Diverse Origins: An Overview from Multiparametric Immunophenotyping to Clinical Applications

Stem cells are known for their capacity to self‐renew and differentiate into at least one specialized cell type. Mesenchymal stem cells (MSCs) were isolated initially from bone marrow but are now known to exist in all vascularized organ or tissue in adults. MSCs are particularly relevant for therapy due to their simplicity of isolation and cultivation. The International Society for Cellular Therapy (ISCT) has proposed a set of standards to define hMSCs for laboratory investigations and preclinical studies: adherence to plastic in standard culture conditions; in vitro differentiation into osteoblasts, adipocytes, and chondroblasts; specific surface antigen expression in which ≥95% of the cells express the antigens recognized by CD105, CD73, and CD90, with the same cells lacking (≤2% positive) the antigens CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA‐DR. In this review we will take an historical overview of how umbilical cord blood, bone marrow, adipose‐derived, placental and amniotic fluid, and menstrual blood stem cells, the major sources of human MSC, can be obtained, identified and how they are being used in clinical trials to cure and treat a very broad range of conditions, including heart, hepatic, and neurodegenerative diseases. An overview of protocols for differentiation into hepatocytes, cardiomyocytes, neuronal, adipose, chondrocytes, and osteoblast cells are highlighted. We also discuss a new source of stem cells, induced pluripotent stem cells (iPS cells) and some pathways, which are common to MSCs in maintaining their pluripotent state.

Evidence for an attentional component of the perceptual misalignment between moving and flashing stimuli

If a pair of dots, diametrically opposed to each other, is flashed in perfect alignment with another pair of dots rotating about the visual fixation point, most observers perceive the rotating dots as being ahead of the flashing dots (flash-lag effect). This psychophysical effect was first interpreted as the result of a perceptual extrapolation of the position of the moving dots. Also, it has been conceived as the result of differential visual latencies between flashing and moving stimuli, arising from purely sensory factors and/or expressing the contribution of attentional mechanisms as well. In a series of two experiments, we had observers judge the relative position between rotating and static dots at the moment a temporal marker was presented in the visual field. In experiment 1 we manipulated the nature of the temporal marker used to prompt the alignment judgment. This resulted in three main findings: (i) the flash-lag effect was observed to depend on the visual eccentricity of the flashing dots; (ii) the magnitude of the flash-lag effect was not dependent on the offset of the flashing dot; and (iii) the moving stimulus, when suddenly turned off, was perceived as lagging behind its disappearance location. Taken altogether, these results suggest that neither visible persistence nor motion extrapolation can account for the perceptual flash-lag phenomenon. The participation of attentional mechanisms was investigated in experiment 2, where the magnitude of the flash-lag effect was measured under both higher and lower predictability of the location of the flashing dot. Since the magnitude of the flash-lag effect significantly increased with decreasing predictability, we conclude that the observers attentional set can modulate the differential latencies determining this perceptual effect. The flash-lag phenomenon can thus be conceived as arising from differential visual latencies which are determined not only by the physical attributes of the stimulus, such as its luminance or eccentricity, but also by attentional mechanisms influencing the delays involved in the perceptual processing.

Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells

Spontaneous Ca(2+) events have been observed in diverse stem cell lines, including carcinoma and mesenchymal stem cells. Interestingly, during cell cycle progression, cells exhibit Ca(2+) transients during the G(1) to S transition, suggesting that these oscillations may play a role in cell cycle progression. We aimed to study the influence of promoting and blocking calcium oscillations in cell proliferation and cell cycle progression, both in neural progenitor and undifferentiated cells. We also identified which calcium stores are required for maintaining these oscillations. Both in neural progenitor and undifferentiated cells calcium oscillations were restricted to the G1/S transition, suggesting a role for these events in progression of the cell cycle. Maintenance of the oscillations required calcium influx only through inositol 1,4,5-triphosphate receptors (IP(3)Rs) and L-type channels in undifferentiated cells, while neural progenitor cells also utilized ryanodine-sensitive stores. Interestingly, promoting calcium oscillations through IP(3)R agonists increased both proliferation and levels of cell cycle regulators such as cyclins A and E. Conversely, blocking calcium events with IP(3)R antagonists had the opposite effect in both undifferentiated and neural progenitor cells. This suggests that calcium events created by IP(3)Rs may be involved in cell cycle progression and proliferation, possibly due to regulation of cyclin levels, both in undifferentiated cells and in neural progenitor cells.

Intracellular Ca2+ Regulation During Neuronal Differentiation of Murine Embryonal Carcinoma and Mesenchymal Stem Cells

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) play a central role in neuronal differentiation. However, Ca(2+) signaling in this process remains poorly understood and it is unknown whether embryonic and adult stem cells share the same signaling pathways. To clarify this issue, neuronal differentiation was analyzed in two cell lines: embryonic P19 carcinoma stem cells (CSCs) and adult murine bone-marrow mesenchymal stem cells (MSC). We studied Ca(2+) release from the endoplasmic reticulum via intracellular ryanodine-sensitive (RyR) and IP(3)-sensitive (IP(3)R) receptors. We observed that caffeine, a RyR agonist, induced a [Ca(2+)](i) response that increased throughout neuronal differentiation. We also demonstrated a functional coupling between RyRs and L- but not with N-, P-, or Q-type Ca(v)1 Ca(2+) channels, both in embryonal CSC and adult MSC. We also found that agonists of L-type channels and of RyRs increase neurogenesis and neuronal differentiation, while antagonists of these channels have the opposite effect. Thus, our data demonstrate that in both cell lines RyRs control internal Ca(2+) release following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels. This study shows that both in embryonal CSC and adult MSC [Ca(2+)](i) is controlled by a common pathway, indicating that coupling of L-type Ca(2+) channels and RyRs may be a conserved mechanism necessary for neuronal differentiation.

Ontogenetic expression of the vanilloid receptors TRPV1 and TRPV2 in the rat retina.

The present study aimed to analyze the gene and protein expression and the pattern of distribution of the vanilloid receptors TRPV1 and TRPV2 in the developing rat retina. During the early phases of development, TRPV1 was found mainly in the neuroblastic layer of the retina and in the pigmented epithelium. In the adult, TRPV1 was found in microglial cells, blood vessels, astrocytes and in neuronal structures, namely synaptic boutons of both retinal plexiform layers, as well as in cell bodies of the inner nuclear layer and the ganglion cell layer. The pattern of distribution of TRPV1 was mainly punctate, and there was higher TRPV1 labeling in the peripheral retina than in central regions. TRPV2 expression was quite distinct. Its expression was virtually undetectable by immunoblotting before P1, and that receptor was found by immunohistochemistry only by postnatal day 15 (P15). RNA and protein analysis showed that the adult levels are only reached by P60, which includes small processes in the retinal plexiform layers, and labeled cellular bodies in the inner nuclear layer and the ganglion cell layer. There was no overlapping between the signal observed for both receptors. In conclusion, our results showed that the patterns of distribution of TRPV1 and TRPV2 are different during the development of the rat retina, suggesting that they have specific roles in both visual processing and in providing specific cues to neural development.

Calcium signaling and cell proliferation.

Cell proliferation is orchestrated through diverse proteins related to calcium (Ca(2+)) signaling inside the cell. Cellular Ca(2+) influx that occurs first by various mechanisms at the plasma membrane, is then followed by absorption of Ca(2+) ions by mitochondria and endoplasmic reticulum, and, finally, there is a connection of calcium stores to the nucleus. Experimental evidence indicates that the fluctuation of Ca(2+) from the endoplasmic reticulum provides a pivotal and physiological role for cell proliferation. Ca(2+) depletion in the endoplasmatic reticulum triggers Ca(2+) influx across the plasma membrane in an phenomenon called store-operated calcium entries (SOCEs). SOCE is activated through a complex interplay between a Ca(2+) sensor, denominated STIM, localized in the endoplasmic reticulum and a Ca(2+) channel at the cell membrane, denominated Orai. The interplay between STIM and Orai proteins with cell membrane receptors and their role in cell proliferation is discussed in this review.

Protecting RNA in fixed tissue: an alternative method for LCM users.

RNA degradation is a major drawback in most common fixation protocols in techniques that require both RNA integrity and preserved morphology, such as laser capture microdissection (LCM) followed by RT-PCR. Moreover, RNA isolation kits especially developed for LCM samples are very expensive. Our aim was to determine an easy protocol that ideally must provide an acceptable morphology, allow proper laser capture of selected cells and improve RNA yield and quality. In this study, retinas were dissected, briefly incubated in a RNA preservative and fixed in 2% paraformaldehyde before being cut on a cryostat. LCM was carried out in retinal sections for immediate RNA isolation, by using TRIzol common protocol with minor modifications. Real-time PCR was performed next in order to compare availability of RNA from samples submitted to different protocols. The use of the RNA preservative followed by a fast fixation did not jeopardize tissue morphology, allowing microdissection of selected cells, combined to minor modifications in usual RNA isolation procedures, significantly improved RNA yield and quality. Furthermore, only LCM samples submitted to our protocol provided amplifiable mRNA, as determined by real-time PCR. Taken together, the combination of the described procedures resulted in a reliable alternative for LCM users.

MicroRNAs in Neuronal Communication

MicroRNAs (miRNAs) are short nucleotides sequences that regulate the expression of genes in different eukaryotic cell types. A tremendous amount of knowledge on miRNAs has rapidly accumulated over the last few years, revealing the growing interest in this field of research. On the other hand, clarifying the physiological regulation of gene expression in the central nervous system is important for establishing a reference for comparison to the diseased state. It is well known that the fine tuning of neuronal networks relies on intricate molecular mechanisms, such as the adjustment of the synaptic transmission. As determined by recent studies, regulation of neuronal interactions by miRNAs has critical consequences in the development, adaptation to ambient demands, and degeneration of the nervous system. In contrast, activation of synaptic receptors triggers downstream signaling cascades that generate a vast array of effects, which includes the regulation of novel genes involved in the control of the miRNA life cycle. In this review, we have examined the hot topics on miRNA gene-regulatory activities in the broad field of neuronal communication-related processes. Furthermore, in addition to indicating the newly described effect of miRNAs on the regulation of specific neurotransmitter systems, we have pointed out how these systems affect the expression, transport, and stability of miRNAs. Moreover, we discuss newly described and under-investigation mechanisms involving the intercellular transfer of miRNAs, aided by exosomes and gap junctions. Thus, in the current review, we were able to highlight recent findings related to miRNAs that indisputably contributed towards the understanding of the nervous system in health and disease.

Blocking of Connexin-Mediated Communication Promotes Neuroprotection during Acute Degeneration Induced by Mechanical Trauma

Accruing evidence indicates that connexin (Cx) channels in the gap junctions (GJ) are involved in neurodegeneration after injury. However, studies using KO animal models endowed apparently contradictory results in relation to the role of coupling in neuroprotection. We analyzed the role of Cx-mediated communication in a focal lesion induced by mechanical trauma of the retina, a model that allows spatial and temporal definition of the lesion with high reproducibility, permitting visualization of the focus, penumbra and adjacent areas. Cx36 and Cx43 exhibited distinct gene expression and protein levels throughout the neurodegeneration progress. Cx36 was observed close to TUNEL-positive nuclei, revealing the presence of this protein surrounding apoptotic cells. The functional role of cell coupling was assessed employing GJ blockers and openers combined with lactate dehydrogenase (LDH) assay, a direct method for evaluating cell death/viability. Carbenoxolone (CBX), a broad-spectrum GJ blocker, reduced LDH release after 4 hours, whereas quinine, a Cx36-channel specific blocker, decreased LDH release as early as 1 hour after lesion. Furthermore, analysis of dying cell distribution confirmed that the use of GJ blockers reduced apoptosis spread. Accordingly, blockade of GJ communication during neurodegeneration with quinine, but not CBX, caused downregulation of initial and effector caspases. To summarize, we observed specific changes in Cx gene expression and protein distribution during the progress of retinal degeneration, indicating the participation of these elements in acute neurodegeneration processes. More importantly, our results revealed that direct control of GJ channels permeability may take part in reliable neuroprotection strategies aimed to rapid, fast treatment of mechanical trauma in the retina.

Prolonged dark adaptation changes connexin expression in the mouse retina

In the retina, ambient light levels influence the cell coupling provided by gap junction (GJ) channels, to compensate the visual function for various lighting conditions. However, the effects of ambient light levels on expression of connexins (Cx), the proteins that form the GJ channels, are poorly understood. In the present study, we first determined whether gene expression of specific Cx (Cx26, Cx31.1, Cx36, Cx37, Cx40, Cx43, Cx45, Cx50, and Cx57) was affected by prolonged dark adaptation. Cx mRNA relative levels were determined in mouse retinas dark adapted for 3 hr, 1 day, and 7 days by using quantitative real‐time PCR. Transcript levels of some Cx were repressed after 3 hr (Cx57), 1 day (Cx45), or 7 days (Cx36 and Cx43) of dark adaptation; others were increased after 1 day (Cx50) or 7 days (Cx31.1 and Cx37); and two of them (Cx26 and Cx40) were not significantly altered. The second aim was to determine whether prolonged dark adaptation affects protein expression of two important Cx in retina: neuronal Cx36 and glial Cx43. We were able to demonstrate that important changes in protein distribution and expression also took place in retina during long‐term dark adaptation. Given their localization, the specific alterations in Cx expression may reflect their distinct response to ambient light levels.