Generation of Functional Human 3D Cortico-Motor Assembloids

May combine two claims.

About 40% of OLIG2+ cells co-express NKX2.2 and about 15% co-express NKX6.1.

“found that ~40% of OLIG2+ cells co-expressed the marker NKX2.2 and ~15% co-expressed NKX6.1”

May combine two claims.

Cortical spheroids form long-range functional connections with hindbrain/cervical spinal cord spheroids, which project and connect with human skeletal muscle.

“Spheroids resembling the cerebral cortex form long-range functional connections with spheroids resembling the hindbrain/cervical spinal cord, and these in turn project and connect with human skeletal muscle.”

May combine two claims.

In day 18 hSpS, OLIG2 is more highly expressed toward the edge, while PAX6 is higher toward the center.

“We found that OLIG2 was more highly expressed toward the edge of the hSpS, whereas PAX6 showed the opposite pattern, with higher expression toward the center”

May combine two claims.

In condition 8 hSpS, SOX1 and PAX6 peaked at day 10, followed by increased OLIG2 and NKX6-1 expression from day 12.

“In condition 8 (hSpS), the neuroectodermal markers SOX1 and PAX6 peaked at day 10, followed by an increase in the expression of OLIG2 and NKX6–1 from day 12”

May combine two claims.

Activity in hSpS-limb assembloids persisted for at least 2 weeks and was completely blocked by curare.

“Activity in hSpS-limb assembloids persisted for at least 2 weeks in vitro and was completely blocked by addition of the acetylcholine receptor antagonist curare (100 µM; χ2 test, p = 0.02”

May combine two claims.

The proportion of active hSkM doubles in hSpS-hSkM compared with hSkM in isolation or co-cultured with hCS, mainly within 1 mm of hSpS, and this effect is blocked by curare.

“we found that the proportion of active hSkM doubled in hSpS-hSkM in comparison to hSkM in isolation or co-cultured with hCS (hSpS-hSkM versus hSkM p = 0.01; Figures 5E and S6D; Video S2, sequence 2). This effect was observed mainly in hSkM within 1 mm of hSpS (hSkM versus hSpS-hSkM <1 mm, p < 0.0001; Figures S6E and S6F) and was blocked by curare”

May combine two claims.

hSpS lacks a clear V3 domain and non-ectodermal cells, but contains an undefined neural group.

“We did not find a clear V3 domain with this analysis, and we found no non-ectodermal cells, however, we identified a group of neural cells that we could not define based on known marker genes.”

May combine two claims.

Optogenetic stimulation at 5 and 8 weeks caused hSkM contraction at both time points, with a decreased success rate at 8 weeks.

“We found that hSkM contracted after light stimulation at both time points, although the success rate for contracting assembloids at 8 weeks tended to be decreased (50% versus 81% success rate at 8 and 5 weeks, respectively; p = 0.06”

May combine two claims.

The eYFP-covered area in hSpS increases progressively over time in hCS-hSpS assembloids, unlike in hCS-hCS assembloids.

“Quantification of the eYFP+-covered area in hSpS in intact hCS-hSpS assembloids showed a progressive increase of eYFP over time (5 daf versus 10 daf p = 0.009; 5 daf versus 20 daf p < 0.0001; Figures 3D, S5F, and S5G for examples of hCS-hSpS assembloids at 20 daf), and this was not observed to the same extent in hCS-hCS assembloids”

May combine two claims.

hSpS expresses HOXB4, HOXC4, and HOXB5.

“we examined the expression of HOX genes in hSpS and found expression of HOXB4, HOXC4, and HOXB5”

May combine two claims.

About 95% of GFP+/mCherry+ cells in hCS co-express MAP2 and fewer than 4% express GFAP.

“We found that ~95% of the GFP+/mCherry+ cells in hCS co-expressed the neuronal marker MAP2 and fewer than 4% expressed the glial lineage marker GFAP”

May combine two claims.

Glutamate uncaging of hCS in hCS-hSpS-hSkM assembloids causes robust muscle contraction, which is blocked by curare and is not related to UV exposure.

“This resulted in robust muscle contraction (p = 0.002) (Figures 6F–6H) suggesting functional assembly of a cortico-spinal-muscle functional unit. The contraction was blocked by treatment with 100 µm curare (Figures 6G, S7D, and S7E) and was not related to UV exposure”

May combine two claims.

Limb buds display spontaneous contractions when assembled with hSpS, but not when kept in isolation or assembled with hCS.

“limb buds displayed spontaneous contractions when assembled with hSpS, but not when kept in isolation or when assembled with hCS (χ2 test, p = 0.02”

• AssumptionRests on an assumption: Mouse limb buds dissected at embryonic day 11.5 have not yet received spinal cord axonal innervation.

May combine two claims.

hSpS contains cholinergic motor neurons, confirmed by immunocytochemistry and electrophysiological recordings.

“We also confirmed the presence of cholinergic motor neurons in hSpS by immunocytochemistry for the enzyme that catalyzes the biosynthesis of the neurotransmitter acetylcholine (CHAT), the vesicular acetylcholine transporter VACHT, the neurofilament SMI-32, and the transcription factors HB9, ISL1, and PHOX2B”

May combine two claims.

Almost 60% of GFP+/mCherry+ cells co-express CTIP2 and about 12% co-express BRN2.

“We found that almost 60% of all GFP+/mCherry+ cells co-expressed the corticofugal marker CTIP2, and only ~12% co-expressed BRN2”

May combine two claims. The quote only partly states this claim.

hCS-derived processes extend into hSpS by 5 days after fusion and form extensive projections by 30 days after fusion.

“Live imaging at 5 days after fusion (daf) showed processes derived from cells in the hCS extending into hSpS”

May combine two claims.

hSpS cells sometimes project into hCS but seldom migrate, and hSpS-derived Hb9 cells did not migrate into hCS.

“hSpS cells sometimes projected into hCS but seldom migrated (Figure S5J). Similarly, we did not observe hSpS-derived Hb9 cells migrating into hCS”

May combine two claims. The quote only partly states this claim.

hSpS contains progenitors, astroglia, dorsal and ventral neuronal domains, and motor neurons expressing ISL1, PRPH, and PHOX2B.

“hSpS cell cluster identity and found they included progenitors expressing SOX9 and TOP2A, astroglia, and several neuronal domains including a dorsal group of cells expressing LHX2, INSM1, ST18, and TLX3”

May combine two claims.

hSpS contains ventral neuronal domains at day 45 and astrocytes and oligodendrocytes at day 75.

“We also observed the presence of ventral neuronal domains in hSpS by immunohistochemistry at day 45 (Figures 2E, 2F, and S4G), as well as the presence of astrocytes and oligodendrocytes at day 75”

May combine two claims.

Retrograde rabies tracing showed extensive GFP expression in hSpS and cells co-expressing GFP and mCherry in hCS.

“We observed extensive expression of GFP in hSpS and cells co-expressing GFP and mCherry in hCS”

May combine two claims.

There is no significant difference in contraction number or covariance between hSpS-hSkM and hCS-hSpS-hSkM assembloids.

“we found no significant difference in the number of contractions or the covariance between hSpS-hSkM and hCS-hSpS-hSkM (p = >0.99)”

May combine two claims.

At 10 weeks, hCS-hSpS-hSkM assembloids contain hCS-derived projections into hSpS, motor neurons, non-reactive astrocytes, myelinating oligodendrocytes, and neuromuscular junctions.

“we used immunohistochemistry in hCS-hSpS-hSkM assembloids at 10 weeks (Figure 7G) and found the presence of hCS-derived projections into hSpS (Figures 7H and S7U), motor neurons (Figure 7I, S7U, and S7V), non-reactive astrocytes (Figures 7H and S7V), myelinating oligodendrocytes (Figures 7H and 7I) and neuromuscular junctions”

May combine two claims.

Optogenetic activation of Chrimson-expressing hCS in hCS-hSpS-hSkM induces global hSkM contractions in 85.7% of tested assembloids and in 40% of pulses.

“Activation of Chrimson-expressing hCS induced global contractions in hSkM in 85.7% of tested assembloids (p = 0.01) (Figures 6L and 6M; Video S5, sequence 1) and 40% of pulses”

May combine two claims.

At 8 weeks after assembly, assembloids contain intact axons and dendrites, mature synapses, organized skeletal muscle fibers, and neuromuscular contacts.

“We found that assembloids at this time present intact axons and dendrites (Figure S7N), mature synapses (Figures 7B, 7C, S7O, and S7P), organized skeletal muscle fibers (Figures 7D and S7Q), and neuromuscular contacts”

May combine two claims.

DAPT exposure from day 19 to 24 in day 45 hSpS causes a roughly 3-fold increase in motor neuron proportion and a 4-fold decrease in an undefined mixed neuronal population, while overall remaining similar to unexposed hSpS.

“When differentiated in the presence of DAPT, although overall similar to the unexposed condition (hSpS(–)DAPT; R2 = 0.85, p < 0.0001), hSpS contained a ~3-fold increase in the proportion of motor neurons as well as a 4-fold decrease in the proportion of an undefined mixed neuronal population”

May combine two claims.

Activation of Chrimson-expressing hCS induces calcium spikes in hSkM fibers, and these responses depend on Chrimson.

“Activation of Chrim-expressing hCS induced calcium spikes in hSkM fibers (Figures 6Q and 6R; Video S5, sequence 2), and these were dependent on the presence of Chrimson”

May combine two claims.

Glutamate uncaging of hSpS in hSpS-hSkM assembloids causes robust hSkM contraction in the presence but not absence of MNI-caged glutamate, and curare stops the contractions.

“Photostimulation of hSpS in hSpS-hSkM assembloids resulted in robust hSkM contraction in the presence, but not in the absence, of MNI-caged glutamate (Figures 5M, 5N, S6O, and S6P; Video S3), and addition of curare stopped hSkM contractions”

May combine two claims.

Differentiated human myotubes express desmin, titin, and heavy chain myosin and become multinucleated.

“Differentiated myotubes expressed markers of mature skeletal muscle, such as desmin (DES), titin (TTN), and the heavy chain myosin (MyHC), and became multinucleated”

May combine two claims.

Light-induced hCS activity elicited calcium spikes in hSkM at both 4-5 and 7-8 weeks, but only the later time point maintained a robust response after five stimulations.

“Light-induced hCS activity elicited calcium spikes in hSkM at both 4–5 and 7–8 weeks, but although hSkM fibers in assembloids respond to the first stimulation at both ages, only at the later time point cells are able to maintain a robust response after 5 stimulations”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Single-cell gene expression profiling of hSpS with and without DAPT recovered 4,175 and 3,173 cells, respectively, with average per-cell values of approximately 17,000 reads, 2,400 molecules, 1,400 genes, and a molecular index coverage of 4.8.

“Gene expression profiles of 4,175 and 3,173 cells were recovered for hSpS with or without DAPT, respectively, with an average number of reads of ~17,000, ~2,400 molecules and ~1,400 number of genes detected per cell with average molecular index coverage (that is, the number of times a molecule was sequenced) of 4.8.”

May combine two claims.

The authors produced a functional human neural circuit from three components that has at least two synaptic connections.

“Third, we showed the generation, from three components, of a functional human neural circuit that contains at least two synaptic contacts.”

Classified as probably not a substantial claim.

Specification of progenitor domains in the hindbrain and spinal cord relies on WNT and retinoic acid along the rostro-caudal axis and sonic hedgehog along the dorso-ventral axis.

“Specification of the progenitor domains in the hindbrain and spinal cord relies on signaling molecules like WNT and retinoic acid (RA) secreted along the rostro-caudal axis, and sonic hedgehog (SHH) along the dorso-ventral axis”

• cites Alaynick et al., 2011 sourceNot checked yet

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• cites Goulding, 2009 sourceNot checked yet

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• cites Jessell, 2000 sourceNot checked yet

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May combine two claims.

The primate cortico-spinal pathway shows species-specific reorganization and direct monosynaptic connections, which may relate to corticalization of motor control and dexterity.

“Recent evidence points at primate-specific reorganization and connectivity of the cortico-spinal pathway, including the presence of direct monosynaptic cortico-spinal connections, which may be related to corticalization of motor control and the evolution of dexterity”

• cites Gu et al., 2017Not checked yet

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• cites Lemon, 2008 sourceNot checked yet

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• cites Sousa et al., 2017 sourceNot checked yet

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May combine two claims.

Long-range neural projections have been observed in cortico-thalamic assembloids, organotypic mouse co-cultures, and sliced cortical organoids with rodent spinal cord explants.

“Long-range neural projections have been observed in a cortico-thalamic assembloid (Xiang et al., 2019), in organotypic mouse co-cultures (Takuma et al., 2002) and in a preparation that used sliced cortical organoids and rodent spinal cord explants (Giandomenico et al., 2019).”

• cites Xiang et al., 2019 sourceNot checked yet

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• cites Takuma et al., 2002 sourceNot checked yet

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• cites Giandomenico et al., 2019 sourceNot checked yet

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May combine two claims.

Components of the cortico-spinal-motor pathway have been generated or studied in isolation, but the in vitro derivation and assembly of this three-part system with human cells had not been achieved.

“Although components of the cortico-spinal-motor pathway have been generated or studied in isolation (Ogura et al., 2018; Sances et al., 2016; Shi et al., 2018; Steinbeck et al., 2016), the in vitro derivation and assembly of this three-part system have not yet been achieved with human cells.”

• cites Ogura et al., 2018 sourceNot checked yet

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• cites Sances et al., 2016 sourceNot checked yet

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• cites Shi et al., 2018 sourceNot checked yet

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May combine two claims.

Cortico-motor assembloids can be used to trace anatomical and functional connectivity, maintained long term, and manipulated optogenetically or pharmacologically to control human muscle contraction.

“these cortico-motor assembloids can be used to trace anatomical and functional connectivity, can be maintained long term in vitro to capture cellular and functional changes, and can be manipulated optogenetically or pharmacologically at multiple levels to control human muscle contraction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Post-mitotic neurons in the spinal cord do not appear to maintain laminar organization at later stages.

“Whereas spinal cord progenitors are organized in a well-defined laminar pattern during development, post-mitotic neurons in the spinal cord do not appear to maintain this organization at later stages”

• cites Lai et al., 2016 sourceNot checked yet

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May combine two claims.

Most hSpS motor neurons express PHOX2B but not TBX20, suggesting a largely somatic identity.

“Most of these cells expressed the motor neuron marker PHOX2B, but did not express the visceral motor neuron marker TBX20, suggesting motor neurons in hSpS have a largely somatic identity”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The balance between V2a interneurons and motor neurons is linked, and loss of motor neurons in Olig1/2 knockout mice increases Chx10+ V2a interneurons.

“The balance between V2a interneurons and motor neurons is known to be linked (Thaler et al., 1999), and loss of motor neurons in Olig1/2 knockout mice results in a dramatic increase of Chx10+ V2a interneurons”

• cites Thaler et al., 1999 sourceNot checked yet

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• cites Zhou and Anderson, 2002Not checked yet

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May combine two claims.

MN cluster 6 is PHOX2B-negative and ISL1/2- and HB9-positive, indicating a limb-innervating lateral motor column-like identity.

“MN cluster 6 was negative for PHOX2B and positive for ISL1/2 and HB9 (MNX1), indicating a limb-innervating lateral motor column-like identity”

Classified as probably not a substantial claim.

The functional output of the cortico-motor circuit is muscle contraction through motor neuron activity.

“The functional output of the cortico-motor circuit is muscle contraction through motor neuron activity.”

May combine two claims.

Some dorsal hSpS clusters were less correlated to mouse spinal cord, possibly because hSpS lacks dorsalizing signals.

“Some dorsal clusters appeared to be less correlated compared to ventral clusters, which may be explained by the lack of dorsalizing signals in hSpS.”

May combine two claims.

Corticofugal projection neurons express CTIP2 and are mainly in deep cortical layers, whereas superficial-layer neurons express BRN2 and project contralaterally.

“Corticofugal projection neurons, which include cortico-spinal projecting cells, express CTIP2 and are mainly located in deep layers of the cerebral cortex, whereas neurons located in superficial layers express BRN2 and project to the contra-lateral cortex”

• cites Greig et al., 2013 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the BD Rhapsody analysis, cells with mitochondrial gene content above 30% were discarded, leaving 7,888 cells from both samples.

“Cells with mitochondrial gene (with a gene symbol starting with MT) content > 30%, were discarded, retaining a total of 7,888 cells from both samples.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Processed gene expression data are deposited in GEO under accession GSE123722.

“Processed gene expression data are available in the Gene Expression Omnibus under accession number GEO: GSE123722.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In the 10x Genomics analysis, cells with more than 6,000 or fewer than 1,000 detected genes, or with mitochondrial content above 18%, were excluded.

“We excluded cells with more than 6,000 or less than 1,000 detected genes, as well as those with a mitochondrial content higher than 18%.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The hiPS cell cultures were tested and kept free of mycoplasma.

“Cultures were tested and maintained mycoplasma free.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

The authors suggest that longer light stimulations may increase success rates.

“Experiments in this manuscript were performed with stimulations of 68–100 ms for increased sampling speed and to avoid any light-induced toxicity, yet longer stimulations may yield higher success rates.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Infecting 3D hSkM with rabies-∆G-Cre-eGFP and AAV-8-CAG-FLEX-Rabies G was difficult, so some assembloids had fewer GFP cells available for quantification.

“Widespread infection of 3D hSkM with rabies-∆G-Cre-eGFP and AAV-8-CAG-FLEX-Rabies G was challenging, which resulted in some assembloids having fewer GFP cells for quantification.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Mouse work was approved by Stanford's animal care and use committee (APLAC).

“Approval for mouse experiments was obtained from the Stanford University’s Administrative Panel on Laboratory Animal Care (APLAC).”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In calcium imaging of hSkM fibers, the numbers of fibers analyzed were 798 for hSkM alone, 652 for hCS + hSkM, 1006 for hSpS + hSkM, and 727 for hSpS + hSkM + curare.

“A total number of 798 (hSkM condition), 652 (hCS + hSkM condition), 1006 (hSpS + hSkM condition) and 727 (hSpS + hSkM + curare condition) hSkM fibers were analyzed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the BD Rhapsody single-cell profiling of hSpS, the proportion of live cells in the pooled samples was estimated to be about 90% using a fluorescent assay.

“the proportion of live cells was estimated using a fluorescent assay (~90%) and all cells were used for further processing.”

Classified as probably not a substantial claim.

The authors had earlier reported that spheroids patterned to specific brain regions offer a modular way to examine human interneuron movement and uncover disease-related phenotypes.

“We previously showed the potential of using region-specific spheroids as a modular approach to study human interneuron migration and identify disease phenotypes (Birey et al., 2017).”

• cites Birey et al., 2017 sourceNot checked yet

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Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Use of the hiPS cell lines received Stanford IRB approval, and all subjects gave informed consent.

“Approval for using these lines was obtained from the Stanford IRB panel and informed consent was obtained from all subjects.”

Classified as probably not a substantial claim.

The hiPS cell lines used were validated by standard methods.

“The hiPS cell lines used in this study were validated using standard methods as previously described (Paşca et al., 2011; Sloan et al., 2018).”

• cites Paşca et al., 2011Not checked yet

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• cites Sloan et al., 2018 sourceNot checked yet

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May combine two claims.

Assembly of intact 3D human cultures resembling the cerebral cortex, hindbrain/spinal cord, and skeletal muscle enables formation of neural circuits that can be manipulated over several weeks to model cortical control of muscle contraction.

“Taken together, these results demonstrate that the assembly of intact 3D human cultures resembling the cerebral cortex, hindbrain/spinal cord and skeletal muscle enables the formation of neural circuits that can be readily manipulated over several weeks in vitro to model cortical control of muscle contraction.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The authors suggest that low opsin expression may lower stimulation success.

“Moreover, because low opsin expression may result in reduced stimulation success, inspection of hCS AAV-1-hSYN1-ChrimsonR-tdT expression before assembly is recommended.”

Classified as probably not a substantial claim.

Human brain specimens were collected under a protocol approved by Stanford's Research Compliance Office.

“Human brain specimens were obtained under a protocol approved by the Research Compliance Office at Stanford University.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The authors state that optogenetic stimulation success depends at least on light-pulse duration and opsin delivery efficiency.

“We note that optogenetic stimulation experiments depend on at least two factors: (i) the duration of light pulses, and (ii) the efficiency of opsin delivery.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The genomic integrity of the hiPS cells was checked with high-density SNP arrays.

“Validation of hiPS genome integrity was performed by high density SNP arrays.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The analysis included only assembloids that had at least 10 GFP-positive cells.

“Only assembloids with at least 10 GFP+ cells were included in the analysis.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the 10x Genomics analysis, genes detected in fewer than three cells were excluded.

“We excluded genes that were not expressed in at least three cells.”

Probably not a substantial claim: may not pass S3 (load-bearing).

In the 10x Genomics analysis, all genes on the X and Y chromosomes were removed from the count matrix because of sex differences between hiPS cell lines.

“Due to sex differences between hiPS cell lines we also removed all genes on the X and Y chromosomes from the count matrix.”