Establishment of single-cell screening system for the rapid identification of transcriptional modulators involved in direct cell reprogramming

Viral screens using focused overexpression libraries can identify putative reprogramming factors, for example, by identifying a defined set of transcription factors that are selectively required to induce pluripotent stem cells or trans-differentiate from mature somatic cell into another mature somatic cell.

“Viral screens using focused overexpression libraries can identify putative reprogramming factors, for example, by identifying a defined set of transcription factors that are selectively required to induce pluripotent stem cells (2–4,7) or trans-differentiate from mature somatic cell into another mature somatic cell (1,8–11).”

• cites [2] Takahashi, 2007, Induction of pluripotent stem cells from adult human fibroblasts by defined factors, Cell sourceNot checked — no full text of the source
• cites [3] Yu, 2007, Induced pluripotent stem cell lines derived from human somatic cells, Science sourceNot checked — no full text of the source
• cites [4] Stadtfeld, 2008, Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse, Cell Stem Cell sourceNot checked — no full text of the source
• cites [7] Takahashi, 2006, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors, Cell sourceNot checked — no full text of the source
• cites [1] Ieda, 2010, Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors, Cell sourceSupported
• cites [8] Zhou, 2008, In vivo reprogramming of adult pancreatic exocrine cells to beta-cells, Nature sourceSupported
• cites [9] Feng, 2008, PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage-like cells, Proc. Natl Acad. Sci. USA sourceNot checked — no full text of the source
• cites [10] Vierbuchen, 2010, Direct conversion of fibroblasts to functional neurons by defined factors, Nature sourcePartly supported

  Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured
• cites [11] Sekiya, 2011, Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors, Nature sourceNot checked — no full text of the source

Pooled screens for cell reprogramming have generally adopted a one-by-one elimination approach or testing for exhaustive combinations and interrogating phenotypes in cell culture from transgenic mice and/or aberrantly cloned cell lines.

“Despite the power of viral technologies, pooled screens for cell reprogramming have generally adopted a one-by-one elimination approach or testing for exhaustive combinations and interrogating phenotypes in cell culture from transgenic mice (1,3,10) and/or aberrantly cloned cell lines (12).”

• cites [1] Ieda, 2010, Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors, Cell sourceSupported
• cites [3] Yu, 2007, Induced pluripotent stem cell lines derived from human somatic cells, Science sourceNot checked — no full text of the source
• cites [10] Vierbuchen, 2010, Direct conversion of fibroblasts to functional neurons by defined factors, Nature sourceSupported
• cites [12] Maherali, 2007, Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution, Cell Stem Cell sourcePartly supported

  The Results (page unavailable) describe screening reprogrammed colonies in culture and studying cloned iPS cell lines. However, the paper focuses on homogeneous, ES-like lines rather than identifying the studied lines as aberrant; it also does not describe a pooled screen using one-by-one elimination or exhaustive factor combinations. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

Genetic interactions are key to defining the transcription regulatory network of human monocytes.

“We and others previously described that the genetic interactions is key to defining the transcription regulatory network of human monocytes (16,17).”

• cites [16] Suzuki, 2009, The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line, Nat. Genet. sourcePartly supported

  The Results section (‘Core transcriptional regulatory network’; page not provided) says that reconstructing the network requires understanding how key regulators’ actions are coordinated, and it describes regulatory edges between them. The study uses the human THP-1 monocytic leukemia cell line, however, rather than human monocytes generally; it describes regulatory interactions, not genetic interactions as such. (LLM, conf=0.82)

  Experimental — accuracy not yet measured
• cites [17] Ravasi, 2010, An atlas of combinatorial transcriptional regulation in mouse and man, Cell sourcePartly supported

  The Results section, “FLI1 and SMAD3 form a heterodimeric complex associated with monocyte development” (page not provided), examines a transcription-factor interaction during differentiation of human THP-1 cells into monocytes. But the paper maps physical interactions between transcription factors, not genetic interactions, and presents the regulatory role of this pair in differentiation as a hypothesis rather than establishing a transcriptional regulatory network of human monocytes. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

HCLS1 has a synergistic role in regulating monocyte-specific gene expressions in dermal fibroblasts in the presence of SPI1.

“We could further demonstrate the synergistic role of HCLS1 in regulating monocyte-specific gene expressions and phagocytosis in dermal fibroblasts in the presence of SPI1.”

In CD14+/HLA-DR+ single cells, SPI1 transcripts were detected in 86/86 cells.

“Highly multiplex Nesc-PCR expression analysis revealed a variety of gene combinations with a significant enrichment of SPI1 (86/86) and a novel transcriptional modulator, HCLS1 (76/86), in the CD14+/HLA-DR+ single cells.”

• Also stated in: § Single-cell gene expression analysis of virus-infected fibroblasts · plain

  “Most notably, single cells positive for CD14 and HLA-DR markers detected SPI1 (86/86, 100%) and 76 out of 86 expressed HCLS1 (88%), which were statistically significant when compared with the M− population where only 8/86 and 10/86 single cells were positive for SPI1 and HCLS1, respectively (chi-square test, P value < 0.001 for both).”

In CD14+/HLA-DR+ single cells, HCLS1 transcripts were detected in 76/86 cells.

“Highly multiplex Nesc-PCR expression analysis revealed a variety of gene combinations with a significant enrichment of SPI1 (86/86) and a novel transcriptional modulator, HCLS1 (76/86), in the CD14+/HLA-DR+ single cells.”

• Also stated in: § Single-cell gene expression analysis of virus-infected fibroblasts · plain

  “Most notably, single cells positive for CD14 and HLA-DR markers detected SPI1 (86/86, 100%) and 76 out of 86 expressed HCLS1 (88%), which were statistically significant when compared with the M− population where only 8/86 and 10/86 single cells were positive for SPI1 and HCLS1, respectively (chi-square test, P value < 0.001 for both).”

HCLS1 has a synergistic role in regulating phagocytosis in dermal fibroblasts in the presence of SPI1.

“We could further demonstrate the synergistic role of HCLS1 in regulating monocyte-specific gene expressions and phagocytosis in dermal fibroblasts in the presence of SPI1.”

The authors established a multiplex single-cell screening system using a fibroblast-to-monocyte transition model to rapidly identify key regulatory factors involved in direct cell reprogramming.

“To rapidly identify key regulatory factors involved in direct cell reprogramming, we established a multiplex single-cell screening system using a fibroblast-to-monocyte transition model.”

• AssumptionRests on an assumption: The stochastic nature of virus infection allows for a mixed combination of transcription modulators to be expressed per infected single cell.

When SPI1 alone was transduced into fibroblasts in an independent experiment, 12 ± 2% of SPI1+ cells expressed the CD14 surface marker.

“In fact, when SPI1 alone was transduced into fibroblasts in an independent experiment, 12 ± 2% of SPI1+ cells expressed the CD14 surface marker suggesting that SPI1 could elicit the expression of this marker but at a low efficiency (Supplementary Figure S3).”

Ablating HCLS1 transcripts in THP1 cells significantly repressed CEBPA expression by 2.56-fold.

“Interestingly, ablating SPI1 and HCLS1 transcripts in THP1 cells could significantly repress IRF8 and CEBPA expression by 2.71-fold and 2.56-fold, respectively (Figure 4D), as well as CSF1R by 6.41-fold—in the case of SPI1 (Figure 4E).”

No HLA-DRB transcript was detected in the nine single cells expressing exogenous SPI1 in the M− population.

“Interestingly, though eight out of nine single cells expressing exogenous SPI1 in the M− population expressed CD14 transcripts, and two out of nine expressed HLA-DRA transcripts, but no detection of HLA-DRB transcript was observed.”

Similar expression patterns were also observed for TYROBP and CD163.

“Similar expression patterns were also observed for TYROBP and CD163.”

Single-cell transduction followed the Poisson distribution.

“Based on our statistical analysis, the single-cell transduction followed the Poisson distribution and the number of detected genes correlated with the half of estimated number of virus particles per cell (Figure 1C).”

A similar expression pattern (augmented CD163 and CD14) was observed for the HCLS1 knockdown.

“Surprisingly, however, THP1 deficient in SPI1 augmented CD163 and CD14 expression, and a similar expression pattern was also observed for the HCLS1 knockdown.”

Rhodamine-positive cells were clearly detected after transducing SPI1 and the SPI1/HCLS1 combination.

“We observed a clear detection of Rhodamine-positive cells after transducing SPI1 and the SPI1/HCLS1 combination.”

Fibroblasts transduced with vector control (10 MOI) failed to express CD14 and HLA-DR surface markers.

“Additionally, fibroblasts transduced with vector control (10 MOI) failed to express CD14 and HLA-DR surface markers (data not shown).”

Eight out of nine single cells expressing exogenous SPI1 in the M− population expressed CD14 transcripts.

“Interestingly, though eight out of nine single cells expressing exogenous SPI1 in the M− population expressed CD14 transcripts, and two out of nine expressed HLA-DRA transcripts, but no detection of HLA-DRB transcript was observed.”

SPI1 alone could induce the expression of CSF1R in dermal fibroblasts.

“Although SPI1 alone could induce the expression of this monocyte marker, the signal intensity was greatly enhanced when fibroblasts were co-infected with HCLS1 (Figure 4A).”

Two weeks post-infection at 10 MOI, 18 ± 2% of the fibroblasts showed Venus fluorescence.

“Two weeks post-infection at 10 MOI, 18 ± 2% of the fibroblasts showed Venus fluorescence (Figure 2A).”

HCLS1 could not enhance the transcript level of CD14.

“However, HCLS1 could not enhance the transcript level of CD14.”

Single cells selected for CD14 and HLA-DR surface proteins revealed 100% detection of CD14, HLA-DRA and HLA-DRB marker transcripts.

“Single cells selected for CD14 and HLA-DR surface proteins revealed 100% detection of CD14, HLA-DRA and HLA-DRB marker transcripts, as expected.”

The number of detected genes correlated with half of the estimated number of virus particles per cell.

“Based on our statistical analysis, the single-cell transduction followed the Poisson distribution and the number of detected genes correlated with the half of estimated number of virus particles per cell (Figure 1C).”

SPI1 and HCLS1 were ubiquitously expressed in all 40 monocyte single cells profiled.

“Interestingly, the single-cell expression profile of primary monocytes revealed that 27% and 22% monocytes expressed CEBPA and IRF8, respectively, which is in contrast to the ubiquitous expression of SPI1 and HCLS1 in all 40 monocyte single cells profiled (Supplementary Figure S2B).”

A minute amount of Venus+ cells were positive for both CD14 and HLA-DR (0.17 ± 0.1%).

“Predictably from a pooled virus, a minute amount of Venus+ cells were positive for both CD14 and HLA-DR (0.17 ± 0.1%; Figure 2B) but was sufficient to obtain single cells for transcript profiling using Nesc-PCR (M+, n = 86).”

HCLS1 greatly enhanced the SPI1-induced expression of CSF1R in dermal fibroblasts.

“Although SPI1 alone could induce the expression of this monocyte marker, the signal intensity was greatly enhanced when fibroblasts were co-infected with HCLS1 (Figure 4A).”

Ablating SPI1 transcripts in THP1 cells significantly repressed CSF1R expression by 6.41-fold.

“Interestingly, ablating SPI1 and HCLS1 transcripts in THP1 cells could significantly repress IRF8 and CEBPA expression by 2.71-fold and 2.56-fold, respectively (Figure 4D), as well as CSF1R by 6.41-fold—in the case of SPI1 (Figure 4E).”

Two out of nine single cells expressing exogenous SPI1 in the M− population expressed HLA-DRA transcripts.

“Interestingly, though eight out of nine single cells expressing exogenous SPI1 in the M− population expressed CD14 transcripts, and two out of nine expressed HLA-DRA transcripts, but no detection of HLA-DRB transcript was observed.”

Cells transduced in combination of SPI1 and HCLS1 ingested 33% more beads when compared with SPI1 alone.

“The image analysis further revealed that cells transduced in combination of SPI1 and HCLS1 ingested 33% more beads when compared with SPI1 alone (Supplementary Figure S5B).”

Hierarchical clustering of single cells reveals distinct cell populations between M+ and M−.

“Hierarchical clustering of single cells reveals distinct cell populations between M+ and M− (A).”

Ablating SPI1 transcripts in THP1 cells significantly repressed IRF8 expression by 2.71-fold.

“Interestingly, ablating SPI1 and HCLS1 transcripts in THP1 cells could significantly repress IRF8 and CEBPA expression by 2.71-fold and 2.56-fold, respectively (Figure 4D), as well as CSF1R by 6.41-fold—in the case of SPI1 (Figure 4E).”

THP1 deficient in SPI1 augmented CD163 and CD14 expression.

“Surprisingly, however, THP1 deficient in SPI1 augmented CD163 and CD14 expression, and a similar expression pattern was also observed for the HCLS1 knockdown.”

More than 50% of the population contained an average number of two to six genes.

“This suggests that a minimum of two copies of viral transcripts can be detected, and 10 MOI of the pooled virus, or 0.55 MOI per gene, was optimal to detect multiple target genes from single cells since >50% of the population contained average number of two to six genes.”

This study establishes a platform for multiplex single-cell screening of combinatorial transcription modulators to drive any direct cell reprogramming.

“This study establishes a platform for a multiplex single-cell screening of combinatorial transcription modulators to drive any direct cell reprogramming.”

HCLS1 is a novel transcriptional modulator.

“a novel transcriptional modulator, HCLS1 (76/86), in the CD14+/HLA-DR+ single cells”

The single-cell 'shotgun-transduction' system enabled prompt detection, isolation and identification of combinatorial genes required to transform fibroblasts into a desired target cell type.

“By relying on the random nature of virus infection, we implemented a single-cell 'shotgun-transduction' system that enabled the prompt detection, isolation and identification of combinatorial genes that are required to transform fibroblasts into a desired target cell type.”

• AssumptionRests on an assumption: The stochastic nature of virus infection allows for a mixed combination of transcription modulators to be expressed per infected single cell.

SPI1 and HCLS1 are a defined set of transcription modulators responsible to switch from human dermal fibroblasts to functional monocyte-like cells.

“By taking advantage of viral transcripts and the single-cell detection method, we noted SPI1 and HCLS1 as a defined set of transcription modulators responsible to switch from human dermal fibroblasts to functional monocyte-like cells.”

The second component narrowly distinguished the monocytes from the M+ cells, indicative of partial (or incomplete) induction of monocyte network in fibroblasts.

“However, a closer examination of the second component narrowly distinguished the monocytes from the M+ cells, indicative of partial (or incomplete) induction of monocyte network in fibroblasts.”

SPI1 alone could induce the expression of CD14 and HLA-DRA in human fibroblasts.

“However, we could additionally demonstrate that CEBPA was dispensable in that SPI1 alone could induce the expression of CD14 and HLA-DRA and promote phagocytosis in human fibroblasts.”

The M+ population, although transcriptionally heterogeneous, was almost exclusively composed of cells expressing high levels of genes characteristic of mature monocytes.

“In a drastic contrast to the M− population, the analysis of M+ population noticeably revealed that this subset, although transcriptionally heterogeneous, was almost exclusively composed of cells expressing high levels of genes characteristic of mature monocytes (Figure 3A).”

The correspondence analysis identified SPI1 and HCLS1 as major determinants to discriminate the monocytes from the original fibroblasts.

“Complementary to PCA, the CoA identified SPI1 and HCLS1 as major determinants to discriminate the monocytes from the original fibroblasts.”

SPI1 alone could induce the expression of CD14.

“Although the sample size was small, the analysis suggests that SPI1 alone could induce the expression of CD14.”

The promotion of monocytic expression and phagocytic function by SPI1 can be greatly enhanced through the co-expression of HCLS1.

“Taken together, our results revealed that SPI1 is minimally required to promote, in part, monocytic expression and phagocytic function in human dermal fibroblasts but can be greatly enhanced through the co-expression of HCLS1.”

SPI1 alone could promote phagocytosis in human fibroblasts.

“However, we could additionally demonstrate that CEBPA was dispensable in that SPI1 alone could induce the expression of CD14 and HLA-DRA and promote phagocytosis in human fibroblasts.”

M+ cells deviated from the original fibroblast state and transitioned into a state comparable to that of CD14+ monocytes.

“These distinct clusters indicate that M+ cells deviated from the original fibroblast state and transitioned into a state comparable to that of CD14+ monocytes.”

The detection of SPI1 by the single-cell screening system decisively validates the method in identifying the strongest modulator in the group.

“The detection of SPI1 by the single-cell screening system decisively validates our method in identifying the strongest modulator in the group.”

A minimum of two copies of viral transcripts can be detected.

“This suggests that a minimum of two copies of viral transcripts can be detected, and 10 MOI of the pooled virus, or 0.55 MOI per gene, was optimal to detect multiple target genes from single cells since >50% of the population contained average number of two to six genes.”

10 MOI of the pooled virus, or 0.55 MOI per gene, was optimal to detect multiple target genes from single cells.

“This suggests that a minimum of two copies of viral transcripts can be detected, and 10 MOI of the pooled virus, or 0.55 MOI per gene, was optimal to detect multiple target genes from single cells since >50% of the population contained average number of two to six genes.”

SPI1 could elicit the expression of CD14 at a low efficiency.

“In fact, when SPI1 alone was transduced into fibroblasts in an independent experiment, 12 ± 2% of SPI1+ cells expressed the CD14 surface marker suggesting that SPI1 could elicit the expression of this marker but at a low efficiency (Supplementary Figure S3).”

The described lentivirus and Nesc-PCR method facilitates rapid identification of key genes involved in trans-differentiation.

“Here we described a rapid and direct method that facilitates the use of lentivirus and Nesc-PCR for identifying key genes involved in trans-differentiation.”

• AssumptionRests on an assumption: The stochastic nature of virus infection allows for a mixed combination of transcription modulators to be expressed per infected single cell.

When the induced expressions were compared with the basal expression of monocytes, the cell transformation was only partial.

“When the induced expressions were compared with the basal expression of monocytes, the cell transformation was only partial (Figure 4B).”

SPI1 is minimally required to promote, in part, monocytic expression and phagocytic function in human dermal fibroblasts.

“Taken together, our results revealed that SPI1 is minimally required to promote, in part, monocytic expression and phagocytic function in human dermal fibroblasts but can be greatly enhanced through the co-expression of HCLS1.”

Our data and published reports strongly suggest that HCLS1 expression correlates positively with enhanced function of monocytes/macrophages possibly via regulating CEBPA expression.

“Although the exact mechanism is unknown, our data and the published reports strongly suggest that HCLS1 expression correlates positively with enhanced function of monocytes/macrophages possibly via regulating CEBPA expression.”

This study demonstrates a rapid single-cell screening workflow to readily identify and characterize new combinatorial factors, applicable to induce various cell-reprogramming pathways.

“Overall, this study demonstrates a rapid single-cell screening workflow to readily identify and characterize new combinatorial factors, applicable to induce various cell-reprogramming pathways.”

This study validated a highly scalable approach for screening lentiviral libraries for direct cell reprogramming.

“In this study, we have validated a highly scalable approach for screening lentiviral libraries for direct cell reprogramming.”