Engineering brain assembloids to interrogate human neural circuits.
The development of neural circuits involves wiring of neurons locally following their generation and migration, as well as establishing long-distance connections between brain regions. Studying these developmental processes in the human nervous system remains difficult because of limited access to tissue that can be maintained as functional over time in vitro. We have previously developed a method to convert human pluripotent stem cells into brain region-specific organoids that can be fused and integrated to form assembloids and study neuronal migration. In contrast to approaches that mix cell lineages in 2D cultures or engineer microchips, assembloids leverage self-organization to enable complex cell-cell interactions, circuit formation and maturation in long-term cultures. In this protocol, we describe approaches to model long-range neuronal connectivity in human brain assembloids. We present how to generate 3D spheroids resembling specific domains of the nervous system and then how to integrate them physically to allow axonal projections and synaptic assembly. In addition, we describe a series of assays including viral labeling and retrograde tracing, 3D live imaging of axon projection and optogenetics combined with calcium imaging and electrophysiological recordings to probe and manipulate the circuits in assembloids. The assays take 3-4 months to complete and require expertise in stem cell culture, imaging and electrophysiology. We anticipate that these approaches will be useful in deciphering human-specific aspects of neural circuit assembly and in modeling neurodevelopmental disorders with patient-derived cells.
View details for DOI 10.1038/s41596-021-00632-z
View details for PubMedID 34992269
Long-term maturation of human cortical organoids matches key early postnatal transitions.
Human stem-cell-derived models provide the promise of accelerating our understanding of brain disorders, but not knowing whether they possess the ability to mature beyond mid- to late-fetal stages potentially limits their utility. We leveraged a directed differentiation protocol to comprehensively assess maturation in vitro. Based on genome-wide analysis of the epigenetic clock and transcriptomics, as well as RNA editing, we observe that three-dimensional human cortical organoids reach postnatal stages between 250 and 300 days, a timeline paralleling in vivo development. We demonstrate the presence of several known developmental milestones, including switches in the histone deacetylase complex and NMDA receptor subunits, which we confirm at the protein and physiological levels. These results suggest that important components of an intrinsic in vivo developmental program persist in vitro. We further map neurodevelopmental and neurodegenerative disease risk genes onto in vitro gene expression trajectories to provide a resource and webtool (Gene Expression in Cortical Organoids, GECO) to guide disease modeling.
View details for DOI 10.1038/s41593-021-00802-y
View details for PubMedID 33619405
Primate cell fusion disentangles gene regulatory divergence in neurodevelopment.
Among primates, humans display a unique trajectory of development that is responsible for the many traits specific to our species. However, the inaccessibility of primary human and chimpanzee tissues has limited our ability to study human evolution. Comparative in vitro approaches using primate-derived induced pluripotent stem cells have begun to reveal species differences on the cellular and molecular levels1,2. In particular, brain organoids have emerged as a promising platform to study primate neural development in vitro3-5, although cross-species comparisons of organoids are complicated by differences in developmental timing and variability of differentiation6,7. Here we develop a new platform to address these limitations by fusing human and chimpanzee induced pluripotent stem cells to generate a panel of tetraploid hybrid stem cells. We applied this approach to study species divergence in cerebral cortical development by differentiating these cells into neural organoids. We found that hybrid organoids provide a controlled system for disentangling cis- and trans-acting gene-expression divergence across cell types and developmental stages, revealing a signature of selection on astrocyte-related genes. In addition, we identified an upregulation of the human somatostatin receptor 2 gene (SSTR2), which regulates neuronal calcium signalling and is associated with neuropsychiatric disorders8,9. We reveal a human-specific response to modulation of SSTR2 function in cortical neurons, underscoring the potential of this platform for elucidating the molecular basis of human evolution.
View details for DOI 10.1038/s41586-021-03343-3
View details for PubMedID 33731928
Chromatin accessibility dynamics in a model of human forebrain development.
Science (New York, N.Y.)
2020; 367 (6476)
Forebrain development is characterized by highly synchronized cellular processes, which, if perturbed, can cause disease. To chart the regulatory activity underlying these events, we generated a map of accessible chromatin in human three-dimensional forebrain organoids. To capture corticogenesis, we sampled glial and neuronal lineages from dorsal or ventral forebrain organoids over 20 months in vitro. Active chromatin regions identified in human primary brain tissue were observed in organoids at different developmental stages. We used this resource to map genetic risk for disease and to explore evolutionary conservation. Moreover, we integrated chromatin accessibility with transcriptomics to identify putative enhancer-gene linkages and transcription factors that regulate human corticogenesis. Overall, this platform brings insights into gene-regulatory dynamics at previously inaccessible stages of human forebrain development, including signatures of neuropsychiatric disorders.
View details for DOI 10.1126/science.aay1645
View details for PubMedID 31974223
Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.
22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.
View details for DOI 10.1038/s41591-020-1043-9
View details for PubMedID 32989314
Reliability of human cortical organoid generation
2019; 16 (1): 75-+
View details for DOI 10.1038/s41592-018-0255-0
View details for Web of Science ID 000454162400033
HEB associates with PRC2 and SMAD2/3 to regulate developmental fates
In embryonic stem cells, extracellular signals are required to derepress developmental promoters to drive lineage specification, but the proteins involved in connecting extrinsic cues to relaxation of chromatin remain unknown. We demonstrate that the helix-loop-helix (HLH) protein, HEB, directly associates with the Polycomb repressive complex 2 (PRC2) at a subset of developmental promoters, including at genes involved in mesoderm and endoderm specification and at the Hox and Fox gene families. While we show that depletion of HEB does not affect mouse ESCs, it does cause premature differentiation after exposure to Activin. Further, we find that HEB deposition at developmental promoters is dependent upon PRC2 and independent of Nodal, whereas HEB association with SMAD2/3 elements is dependent of Nodal, but independent of PRC2. We suggest that HEB is a fundamental link between Nodal signalling, the derepression of a specific class of poised promoters during differentiation, and lineage specification in mouse ESCs.
View details for DOI 10.1038/ncomms7546
View details for Web of Science ID 000352720200002
View details for PubMedID 25775035
Function of COP9 Signalosome in Regulation of Mouse Oocytes Meiosis by Regulating MPF Activity and Securing Degradation
2011; 6 (10)
The COP9 (constitutive photomorphogenic) signalosome (CSN), composed of eight subunits, is a highly conserved protein complex that regulates processes such as cell cycle progression and kinase signalling. Previously, we found the expression of the COP9 constitutive photomorphogenic homolog subunit 3 (CSN3) and subunit 5 (CSN5) changes as oocytes mature for the first time, and there is no report regarding roles of COP9 in the mammalian oocytes. Therefore, in the present study, we examined the effects of RNA interference (RNAi)-mediated transient knockdown of each subunit on the meiotic cell cycle in mice oocytes. Following knockdown of either CSN3 or CSN5, oocytes failed to complete meiosis I. These arrested oocytes exhibited a disrupted meiotic spindle and misarranged chromosomes. Moreover, down-regulation of each subunit disrupted the activity of maturation-promoting factor (MPF) and concurrently reduced degradation of the anaphase-promoting complex/cyclosome (APC/C) substrates Cyclin B1 and Securin. Our data suggest that the CSN3 and CSN5 are involved in oocyte meiosis by regulating degradation of Cyclin B1 and Securin via APC/C.
View details for DOI 10.1371/journal.pone.0025870
View details for Web of Science ID 000295943000088
View details for PubMedID 21991377
View details for PubMedCentralID PMC3185060
Chromatin and transcriptional signatures for Nodal signaling during endoderm formation in hESCs
2011; 357 (2): 492-504
The first stages of embryonic differentiation are driven by signaling pathways hardwired to induce particular fates. Endoderm commitment is controlled by the TGF-β superfamily member, Nodal, which utilizes the transcription factors, SMAD2/3, SMAD4 and FOXH1, to drive target gene expression. While the role of Nodal is well defined within the context of endoderm commitment, mechanistically it is unknown how this signal interacts with chromatin on a genome wide scale to trigger downstream responses. To elucidate the Nodal transcriptional network that governs endoderm formation, we used ChIP-seq to identify genomic targets for SMAD2/3, SMAD3, SMAD4, FOXH1 and the active and repressive chromatin marks, H3K4me3 and H3K27me3, in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that while SMAD2/3, SMAD4 and FOXH1 associate with DNA in a highly dynamic fashion, there is an optimal bivalent signature at 32 gene loci for driving endoderm commitment. Initially, this signature is marked by both H3K4me3 and H3K27me3 as a very broad bivalent domain in hESCs. Within the first 24h, SMAD2/3 accumulation coincides with H3K27me3 reduction so that these loci become monovalent marked by H3K4me3. JMJD3, a histone demethylase, is simultaneously recruited to these promoters, suggesting a conservation of mechanism at multiple promoters genome-wide. The correlation between SMAD2/3 binding, monovalent formation and transcriptional activation suggests a mechanism by which SMAD proteins coordinate with chromatin at critical promoters to drive endoderm specification.
View details for DOI 10.1016/j.ydbio.2011.06.009
View details for Web of Science ID 000294834400019
View details for PubMedID 21741376
HEB and E2A function as SMAD/FOXH1 cofactors
GENES & DEVELOPMENT
2011; 25 (15): 1654-1661
Nodal signaling, mediated through SMAD transcription factors, is necessary for pluripotency maintenance and endoderm commitment. We identified a new motif, termed SMAD complex-associated (SCA), that is bound by SMAD2/3/4 and FOXH1 in human embryonic stem cells (hESCs) and derived endoderm. We demonstrate that two basic helix-loop-helix (bHLH) proteins-HEB and E2A-bind the SCA motif at regions overlapping SMAD2/3 and FOXH1. Furthermore, we show that HEB and E2A associate with SMAD2/3 and FOXH1, suggesting they form a complex at critical target regions. This association is biologically important, as E2A is critical for mesendoderm specification, gastrulation, and Nodal signal transduction in Xenopus tropicalis embryos. Taken together, E proteins are novel Nodal signaling cofactors that associate with SMAD2/3 and FOXH1 and are necessary for mesendoderm differentiation.
View details for DOI 10.1101/gad.16800511
View details for Web of Science ID 000293700900011
View details for PubMedID 21828274
View details for PubMedCentralID PMC3182016
Role of Bcl2-like 10 (Bcl2l10) in Regulating Mouse Oocyte Maturation
BIOLOGY OF REPRODUCTION
2009; 81 (3): 497-506
Previously, we have shown that Bcl2l10 is highly expressed in metaphase II (MII)-stage oocytes. The objective of this study was to characterize Bcl2l10 expression in ovaries and to examine the function of Bcl2l10 in oocyte maturation using RNA interference. Bcl2l10 transcript expression was ovary and oocyte specific. Bcl2l10 was highly expressed in oocytes and pronuclear-stage embryos; however, its expression decreased at the two-cell stage and dramatically disappeared thereafter. Microinjection of Bcl2l10 double-stranded RNA into the cytoplasm of germinal vesicle oocytes resulted in a marked decrease in Bcl2l10 mRNA and protein and metaphase I (MI) arrest (78.9%). Most MI-arrested oocytes exhibited abnormalities in their spindles and chromosome configurations. Bcl2l10 RNA interference had an obvious effect on the activity of maturation-promoting factor but not on that of mitogen-activated protein kinase. We concluded that the role of Bcl2l10 is strongly associated with oocyte maturation, especially at the MI-MII transition.
View details for DOI 10.1095/biolreprod.108.073759
View details for Web of Science ID 000269256400007
View details for PubMedID 19439730
Role of cytosolic malate dehydrogenase in oocyte maturation and embryo development
Fertility and sterility
2006; 86 (4): 1129-1136
View details for DOI 10.1016/j.fertnstert.2006.02.105
Gene expression profiling of early follicular development in primordial, primary, and secondary follicles
Fertility and sterility
2006; 85 (1): 193-203
View details for DOI 10.1016/j.fertnstert.2006.02.105
Identification of differential gene expression in germinal vesicle vs. metaphase II mouse oocytes by using annealing control primers
FERTILITY AND STERILITY
2005; 83: 1293-1296
By using a new innovative technology, annealing control primer-polymerase chain reaction (ACP-PCR), we identified genes that are differentially expressed in immature GV vs. in mature MII mouse oocytes. The results of the present study will be valuable in understanding the components of oocyte maturation and provide a basis for studies of human oocyte maturation.
View details for DOI 10.1016/j.fertnstert.2004.09.037
View details for Web of Science ID 000228526600027
View details for PubMedID 15831304
Leptin receptors are down-regulated in uterine implantation sites compared to interimplantation sites
MOLECULAR AND CELLULAR ENDOCRINOLOGY
2005; 232 (1-2): 27-35
Leptin is a circulating hormone that plays an important role in the regulation of metabolism, obesity, and reproduction. Leptin binds to its receptors on the cell membrane and is involved in the activation of STAT3. Recently, endometrium was suggested to be a novel target for leptin recently. We, therefore, examined the expression of leptin, leptin receptors, and STAT3 in the mouse uterus (implantation and interimplantation sites) to investigate the role of the leptin system during the early implantation period. Leptin mRNA was not detected in mouse uterine tissues or blastocysts, although adipose tissue, the positive control, showed a strong signal. Both of the receptor splice variants were expressed in the uterus and blastocysts, but the mRNA level was much lower in implantation sites compared to interimplantation sites. The mRNA expression of leptin receptors was determined to be higher in stromal cells than in the luminal epithelium using laser capture microdissection (LCM) analysis. Using immunohistochemistry, leptin was detected as a strong signal in the luminal epithelium and embryo, whereas the receptor was detected in subepithelial stromal cells rather than the luminal epithelium. As leptin itself was not detected by RT-PCR, the immunohistologically detected leptin may originate elsewhere, such as in adipose tissue. The differential expression of leptin receptors in implantation sites compared to interimplantation sites suggests that the leptin/leptin receptor system may be a delicate regulator of the implantation process.
View details for DOI 10.1016/j.mce.2005.01.002
View details for Web of Science ID 000227758500004
View details for PubMedID 15737466
Effects of bone morphogenetic protein-7 (BMP-7) on primordial follicular growth in the mouse ovary
MOLECULAR REPRODUCTION AND DEVELOPMENT
2004; 69 (2): 159-163
Previously, bone morphogenetic protein-7 (BMP-7) was suggested as a factor that may act to facilitate the transition of follicles from primordial stage to the pool of developed primary, preantral, and antral follicles (Lee et al. 2001: Biol Reprod 65:994-999.). Thus, aim of the present study was to evaluate effect(s) of BMP-7 on the primordial-primary follicle transition. Neonatal mouse ovaries were cultured in the presence or absence of 100 mIU/ml FSH with various doses of BMP-7 (0, 10, and 100 ng/ml). After 4-day culture period, number of follicles was counted and the expression of transcripts for FSH receptor (FSHR), kit ligand (KL), and c-kit was measured by RT-PCR. BMP-7 alone at 100 ng/ml concentration stimulated follicle development with concurrent increase of mRNA for FSHR. BMP-7 alone down-regulated KL expression however, the ratio between KL1 and KL2 was increased. There was no change in the c-kit mRNA expression. Results of the present study suggest that the BMP-7 is one of the factors involved in primordial-primary follicle transition in the mouse ovary and it may play a role in expression of FSHR for further follicular development.
View details for DOI 10.1002/mrd.20163
View details for Web of Science ID 000223711100006
View details for PubMedID 15293217
A molecular basis for embryo apposition at the luminal epithelium
MOLECULAR AND CELLULAR ENDOCRINOLOGY
2004; 219 (1-2): 95-104
To obtain a gene expression profile during embryo apposition to the luminal epithelium, we isolated mouse luminal epithelium from implantation (IM) and interimplantation (INTER) sites using laser capture microdissection (LCM), and analyzed their gene expression by microarray analysis. IM and INTER sites were sampled on day 4.5 after mating of female mice with fertile males (day 0.5 = vaginal plug). RNA was extracted, amplified, labeled, and hybridized to microarrays and results were analyzed using the significance analysis of microarrays (SAM) method. Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes most highly expressed at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were ESTs or were of unknown function, and the remain 53 genes had known functions mainly relating to cellular structuring and others such as cell cycling, gene/protein expression, immune responses, invasion, metabolism, oxidative stress, or signal transduction. Specifically, of the 24 structural genes, 14 were implicated in extracellular matrix and tissue remodeling. Meanwhile, of the 13 genes that were highly expressed at INTER, eight were ESTs or of unknown function, and the remaining five were implicated in metabolism, signal transduction, and gene/protein expression. Among these 58 (53 + 5) genes with known functions, 13 genes (22.4%) were associated with Ca2+ for their function. Results of the present study suggest that (1) at IM sites, active tissue remodeling is occurring for embryo invasion while the INTER sites are relatively quiescent and (2) Ca2+ may be a vital regulatory factor in the apposition process. Investigations of human homologues of those genes expressed in the mouse luminal epithelium during apposition may help to understand the implantation process and/or implantation failure in humans.
View details for DOI 10.1016/j.mce.2004.01.007
View details for Web of Science ID 000221835500011
View details for PubMedID 15149731
Nitric oxide-mediated inhibition of follicular apoptosis is associated with HSP70 induction and Bax suppression
MOLECULAR REPRODUCTION AND DEVELOPMENT
2002; 61 (4): 504-510
Nitric oxide (NO) has recently emerged as a potential regulator of follicular development because of its involvement in the regulation of several physiological functions of the ovary. NO influences apoptotic cell death of follicular cells as a follicle survival factor. The present study was conducted (1) to investigate the mechanism involved in the protective effect of NO on spontaneously induced follicular apoptosis in serum-free condition and (2) to determine the role of NO on the expression of mRNAs and proteins for HSP70 and Bax. Preovulatory follicles obtained from PMSG-primed rats were cultured for 24 hr in serum-free medium with or without sodium nitroprusside (SNP), a NO generator. Granulosa cells within follicles incubated in medium alone for 24 hr exhibited extensive apoptosis. Treatment of SNP in the culture medium blocked this onset of apoptosis. Both mRNA and protein levels of HSP70 were highly increased with SNP than those of control group. On the contrary, those of Bax were suppressed with SNP treatment. Results of the present study suggest that NO prevents rat preovulatory follicular apoptosis in vitro by stimulating HSP70 and suppressing Bax expression.
View details for DOI 10.1002/mrd.10033
View details for Web of Science ID 000174157900010
View details for PubMedID 11891922