the role of gravity mechanotransduction in …ebs, of genes associated with glycolysis (slc2a1,...

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Single-cell RNA-seq technologies are revolutionizing the field of molecular biology, providing the capacity to go beyond global gradation analysis of gene activity to single cell expression within a population. Our results suggest that gravity mechanostimulation elicits increased cell proliferation and differentiation, and that these effects at the single cell expressome level are most notable on a specific cell-cycle clusters. THE ROLE OF GRAVITY MECHANOTRANSDUCTION IN REGULATING STEM CELL TISSUE REGENERATIVE POTENTIAL AT THE SINGLE CELL EXPRESSOME LEVEL Cassandra M. Juran 1,2 , Justina Žvirblytė 3,4 , Molly Coyne 1 , Eduardo A.C. Almeida 1 1 Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA 2 Universities Space Research Association, Mountain View, CA 3 Life Sciences Center, Vilnius University, Vilnius, Lithuania 4 International Internship (I 2 ) Program Methods 1.Blaber, Elizabeth A et al. “Microgravity Reduces the Differentiation and Regenerative Potential of Embryonic Stem Cells” Stem cells and development vol. 24,22 (2015): 2605-21. Gravity is an omnipresent force on Earth, and all living organisms have evolved under the influence of constant gravity. Mechanical forces generated by gravity are potent modulators of stem cell based tissue regenerative mechanisms, inducing cell fate decisions and tissue specific commitment. A novel mechanical unloading investigation assessed the formation, morphology, and gene expression of embryoid bodies (EB), a transitory cell model of early differentiation. After 15 days of spaceflight, the mechanotransduction-null EB cells showed upregulated proliferative mechanisms while differentiation cues were silenced (Fig. 2). Clonally-derived ESCs were plated on a collagen matrix and expanded for 36 hours. EBs were aggregated from ESCs. After formation, the EBs were transferred to a collagen matrix culture dish and given 4 days to allow implantation and outgrowth. Both cultures were then subjected to either a 60 min 50g centrifugal pulse of gravity mechanotransduction, or no stimulation. Six hours post-stimulation, we used a 10X Genomics Chromium/Single Cell controller to generate bar-coded single cell Illumina libraries and sequenced ESC and EB expressome libraries for 5,000 1g control cells, and 5,000 cells pulse-stimulated with 50g, simulating running/jumping bone marrow hydrostatic pressures Results – Aggregated Libraries Expression of key genes associated with pluripotency, lineage commitment, and cell cycle Figure 3. t-SNE visualization of individual mESCs and EBs to identify subpopulations. Colors denote corresponding clusters.. Figure 2. Mouse embryonic stem cells differentiated into embryoid bodies (EBs) and exposed to spaceflight for 15 days, exhibited significant down-regulation of multiple terminal lineage markers. Specifically, over 90% of genes expressed in ground controls related to differentiation of terminal lineages indicating that microgravity suppresses stem cell differentiation and regeneration capacity [1] Gravity mechanotransduction regulates stem cell tissue regenerative processes by modulating stem cell proliferation and differentiation fates at specific cell cycle stages Hypothesis Results – Mechanoregulation Background and Experimental Rationale Conclusion Figure 1. Embryonic Stem Cells (panels A and B) are a fundamental proliferative stem cell model for study of development and regeneration research. Embryonic Stem Cell derived Embryoid Bodies (panels C and D) serve as a model of complex differentiation pathways, recapitulating a normal embryo development in utero. Fluorescent staining of cells. Blue – DAPI, nucleus; green – actin, cytoskeleton. (A) ESCs imaged using phase contrast at 20X and (B) ESCs florescent labeled at 40X. (C) Brightfield EBs at 4X and (D) EBs florescent labeled at 10X. A B C D = stim reverts EB to ESC = universally mechanoregulated = suppression in EB-stim = increased activity in EB-stim The segregation of expression profiles is most evident between the ESC culture and EB cultures. Comparatively, expression changes due to mechanical regulation are less impactful; however, EBs demonstrate significant mechanotransduction in genes related to cell motility, ECM development and proliferation related chromosome condensation. Interestingly, mechanical stimulation of the early differentiation EBs depresses several genes back toward ESC levels (Fig 4 box 2). The majority of these genes are related to proliferative regulation and glycolysis metabolic regulation. Mechanical stimulation in the EBs causes suppression, compared to unstimulated EBs, of genes associated with glycolysis (Slc2a1, Aloda, Ldha, Gpi1, and Pkm) while simultaneously increasing the expression of genes associated with mitochondrial oxidative phosphorylation and the electron transport chain (mt- Co3, mt-Co1, and Sdhc). Pgk1 – glycolysis enzyme Mechanoregulated suppression in EB cells Cluster distribution of the individual libraries generated by single cell sequencing illustrate heterogeneity and subpopulations within the cultured cells (Fig. 3). Clusters are generated by the graph-based hierarchical method. Clustering identifies like cell by expression profiling Our cursory assessment identified cluster specific cells positive for Nanog, SCF, Oct4, Sox2 and PCNA within the ESC and EB cultures. Slc2a1, Aldoa, Ldha, Wfdc2, Gpi1, Galk1, Igfbp2, Pold2, Blvrb, Fam162a, Gm42418, Fdps, Pkm Ftl1-psi, Rps27rt, Fn1, rnt-C03, Bri3, Cfap20, H3t3b, rnt-Co1, Hmmr, Sdnc, Top2a, Vbp1, Dppa3, Tra2a, Cltc EB_HG EB_null ESC_HG ESC_null Pgk1 Car4 Hmgn5 Upp1, Slc16a3 Jund, Erh Sms, Plk4 1 2 3 4 5 6 7 EB_HG EB_null ESC_HG ESC_null Stem cell specific and cell signaling markers Stem cell and related signaling markers are inversely expressed after commitment to early differentiation (ESC -> EB). Mechanical loading regulates several genes involved in signaling induction of embryogenesis folding (Wnt1) and differentiation (Foxa2, Actc1, Ccnd2, krt15). Marker of Pluripotency p53 and cell cycle regulation markers Early lineage commitment Terminal differentiation markers Cell cycle inhibitors (TRP53, CDKN1a, CDKN2a) are suppressed by mechanical stimulation. This suppression lessens mitotic restrictions and encourages proliferation. Mechanical stimulation in differentiation committed cells upregulates terminal markers. However, single cell sequencing reveals that with mechanical stimulation a sub-population of EB cells returns to expression profiles representative of a earlier pluripotent state. Fig. 5. Lefty2 expression profile for all 4 conditions. Fig. 6. TRP53 expression Figure 7. Myc expression. Heatmap of genes associated with Stem Cell Pluripotency, Lineage Differentiation, and the Cell Cycle Pluripotency is reduced with commitment to early differentiation (ESC->EB). Mechanical stimulation in EB depresses genes associated with exit from the pluripotent cell state; returning gene expression toward those of undifferentiated ESCs. Mechanical stimulation only encourages increased expression of early lineage commitment markers in the EB cells. Indicating that mechanical stimulation can only influence differentiation commitment after transition from totipotency. 4 0 -4 Figure 4. Heatmap of most significantly up- and don-regulated genes between the 4 conditions. Interesting for many of the same genes the opposite regulation is seen for the mechanostimulated ESCs compared to unstimulated ESCs (Fig.4inserts 1, 5, 6, and 7). Pgk1, a gene responsible for conversion of glucose to pyruvate upon entering cellular respiration, has elevated population expression in the early differentiation EBs compared to the ESCs. Mechanical stimulation depressed Pgk1 in the EB cells. Glycolysis related genes Oxidative phosphorylation, motility, and pluripotency related genes Mitosis related genes Histone compaction Figure 5. Pgk1 population relative expression for all 4 groups . https://ntrs.nasa.gov/search.jsp?R=20190027258 2020-06-02T03:01:26+00:00Z

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Page 1: THE ROLE OF GRAVITY MECHANOTRANSDUCTION IN …EBs, of genes associated with glycolysis (Slc2a1, Aloda, Ldha, Gpi1, and Pkm) while simultaneously increasing the expression of genes

Single-cell RNA-seq technologies are revolutionizing the field of molecular biology, providing the capacity to go beyond global gradation analysis of gene

activity to single cell expression within a population.Our results suggest that gravity mechanostimulation elicits increased cell proliferation and differentiation, and that these effects at the single cell

expressome level are most notable on a specific cell-cycle clusters.

THE ROLE OF GRAVITY MECHANOTRANSDUCTION IN REGULATING STEM CELL TISSUE REGENERATIVE POTENTIAL AT THE SINGLE CELL EXPRESSOME LEVEL

Cassandra M. Juran1,2, Justina Žvirblytė3,4 , Molly Coyne1, Eduardo A.C. Almeida1 1Space Biosciences Division, NASA Ames Research Center, Moffett Field, CA

2Universities Space Research Association, Mountain View, CA3Life Sciences Center, Vilnius University, Vilnius, Lithuania

4International Internship (I2) Program

Methods

1.Blaber, Elizabeth A et al. “Microgravity Reduces the Differentiation and Regenerative Potential of Embryonic Stem Cells” Stem cells and development vol. 24,22 (2015): 2605-21.

• Gravity is an omnipresent force on Earth, and all living organisms have evolvedunder the influence of constant gravity.

• Mechanical forces generated by gravity are potent modulators of stem cell basedtissue regenerative mechanisms, inducing cell fate decisions and tissue specificcommitment.

• A novel mechanical unloading investigation assessed the formation, morphology,and gene expression of embryoid bodies (EB), a transitory cell model of earlydifferentiation. After 15 days of spaceflight, the mechanotransduction-null EBcells showed upregulated proliferative mechanisms while differentiation cueswere silenced (Fig. 2).

• Clonally-derived ESCs were plated on a collagen matrix and expanded for 36 hours.• EBs were aggregated from ESCs. After formation, the EBs were transferred to a

collagen matrix culture dish and given 4 days to allow implantation and outgrowth.• Both cultures were then subjected to either a 60 min 50g centrifugal pulse of gravity

mechanotransduction, or no stimulation.• Six hours post-stimulation, we used a 10X Genomics Chromium/Single Cell controller

to generate bar-coded single cell Illumina libraries and sequenced ESC and EBexpressome libraries for 5,000 1g control cells, and 5,000 cells pulse-stimulated with50g, simulating running/jumping bone marrow hydrostatic pressures

Results – Aggregated LibrariesExpression of key genes associated with

pluripotency, lineage commitment, and cell cycle

Figure 3. t-SNE visualization of individual mESCs andEBs to identify subpopulations. Colors denotecorresponding clusters..

Figure 2. Mouse embryonic stem cells differentiated into embryoid bodies (EBs) and exposed to spaceflight for 15 days, exhibited significant down-regulation of multiple terminal lineage markers. Specifically, over 90% of genes expressed in ground controls related to differentiation of terminal lineages indicating that microgravity suppresses stem cell differentiation and regeneration capacity [1]

Gravity mechanotransduction regulates stem cell tissue regenerative processes by modulating stem cell proliferation and

differentiation fates at specific cell cycle stages

Hypothesis

Results – Mechanoregulation Background and Experimental Rationale

Conclusion

Figure 1. Embryonic Stem Cells (panels A and B) are a fundamental proliferative stem cell model for study of development and regeneration research. Embryonic Stem Cell derived Embryoid Bodies (panels C and D) serve as a model of complex differentiation pathways, recapitulating a normal embryo development in utero. Fluorescent staining of cells. Blue – DAPI, nucleus; green – actin, cytoskeleton. (A) ESCsimaged using phase contrast at 20X and (B) ESCs florescent labeled at 40X. (C) Brightfield EBs at 4X and (D) EBs florescent labeled at 10X.

A B

C D

= stim reverts EB to ESC= universally mechanoregulated = suppression in EB-stim= increased activity in EB-stim

• The segregation of expression profiles is most evident between the ESC culture andEB cultures. Comparatively, expression changes due to mechanical regulation areless impactful; however, EBs demonstrate significant mechanotransduction ingenes related to cell motility, ECM development and proliferation relatedchromosome condensation.

• Interestingly, mechanical stimulation of the early differentiation EBs depressesseveral genes back toward ESC levels (Fig 4 box 2). The majority of these genes arerelated to proliferative regulation and glycolysis metabolic regulation.

• Mechanical stimulation in the EBs causes suppression, compared to unstimulatedEBs, of genes associated with glycolysis (Slc2a1, Aloda, Ldha, Gpi1, and Pkm)while simultaneously increasing the expression of genes associated withmitochondrial oxidative phosphorylation and the electron transport chain (mt-Co3, mt-Co1, and Sdhc).

Pgk1 – glycolysis enzymeMechanoregulated suppression in EB cells

• Cluster distribution of theindividual libraries generatedby single cell sequencingillustrate heterogeneity andsubpopulations within thecultured cells (Fig. 3).

• Clusters are generated by thegraph-based hierarchicalmethod.

• Clustering identifies like cell byexpression profiling

• Our cursory assessmentidentified cluster specific cellspositive for Nanog, SCF, Oct4,Sox2 and PCNA within the ESCand EB cultures.

Slc2a1, Aldoa, Ldha, Wfdc2, Gpi1, Galk1, Igfbp2, Pold2, Blvrb, Fam162a, Gm42418, Fdps, Pkm

Ftl1-psi, Rps27rt, Fn1, rnt-C03, Bri3, Cfap20, H3t3b, rnt-Co1, Hmmr, Sdnc, Top2a, Vbp1, Dppa3, Tra2a, Cltc

EB_HG

EB_null

ESC_HG

ESC_null

Pgk1 Car4

Hmgn5

Upp1, Slc16a3

Jund, Erh

Sms, Plk4

1

23

4

5 67

EB_HG EB_null ESC_HG ESC_null

Stem cell specific and cell signaling markersStem cell and related signaling markers are inversely expressedafter commitment to early differentiation (ESC -> EB).Mechanical loading regulates several genes involved in signalinginduction of embryogenesis folding (Wnt1) and differentiation(Foxa2, Actc1, Ccnd2, krt15). Marker of Pluripotency

p53 and cell cycle regulation markers

Early lineage commitment

Terminal differentiation markers

• Cell cycle inhibitors (TRP53,CDKN1a, CDKN2a) are suppressedby mechanical stimulation. Thissuppression lessens mitoticrestrictions and encouragesproliferation.

Mechanical stimulation indifferentiation committed cellsupregulates terminal markers.However, single cell sequencingreveals that with mechanicalstimulation a sub-population ofEB cells returns to expressionprofiles representative of aearlier pluripotent state.

Fig. 5. Lefty2 expressionprofile for all 4 conditions.

Fig. 6. TRP53 expression

Figure 7. Myc expression.

Heatmap of genes associated with Stem Cell Pluripotency, Lineage Differentiation, and the Cell Cycle

• Pluripotency is reduced withcommitment to earlydifferentiation (ESC->EB).

• Mechanical stimulation in EBdepresses genes associated withexit from the pluripotent cell state;returning gene expression towardthose of undifferentiated ESCs.

• Mechanical stimulation only encourages increasedexpression of early lineage commitment markers in theEB cells. Indicating that mechanical stimulation canonly influence differentiation commitment aftertransition from totipotency.

4 0 -4

Figure 4. Heatmap of most significantly up- and don-regulated genes between the 4 conditions.

• Interesting for many of the samegenes the opposite regulation isseen for the mechanostimulatedESCs compared to unstimulatedESCs (Fig.4inserts 1, 5, 6, and 7).

• Pgk1, a gene responsible forconversion of glucose to pyruvateupon entering cellular respiration,has elevated population expressionin the early differentiation EBscompared to the ESCs. Mechanicalstimulation depressed Pgk1 in theEB cells.

Glycolysis related genesOxidative phosphorylation, motility, and pluripotency

related genes

Mitosis related genes

Histone compaction

Figure 5. Pgk1 population relative expressionfor all 4 groups .

https://ntrs.nasa.gov/search.jsp?R=20190027258 2020-06-02T03:01:26+00:00Z