Integrated transcriptomic analysis of human induced pluripotent stem cell-derived osteogenic differentiation reveals a regulatory role of KLF16
Curation statements for this article:-
Curated by eLife
eLife Assessment
The authors investigated KLF Transcription Factor 16 (KLF16) as an inhibitor of osteogenic differentiation, which plays a critical role in bone development, metabolism and repair. The results of the study are valuable as they could help to facilitate future research on the regulation of osteogenesis in vitro and in vivo. However, the evidence overall is incomplete, as validation by knockout mouse models would help to strengthen the conclusions.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Abstract
Osteogenic differentiation is essential for bone development, metabolism, and repair; however, the underlying regulatory relationships among genes remain poorly understood. To elucidate the transcriptomic changes and identify novel regulatory genes involved in osteogenic differentiation, we differentiated mesenchymal stem cells (MSCs) derived from 20 human iPSC lines into preosteoblasts (preOBs) and osteoblasts (OBs). We then performed transcriptome profiling of MSCs, preOBs and OBs. The iPSC-derived MSCs and OBs showed similar transcriptome profiles to those of primary human MSCs and OBs, respectively. Differential gene expression analysis revealed global changes in the transcriptomes from MSCs to preOBs, and then to OBs, including the differential expression of 840 genes encoding transcription factors (TFs). TF regulatory network analysis uncovered a network comprising 451 TFs, organized into five interactive modules. Multiscale embedded gene co-expression network analysis (MEGENA) identified gene co-expression modules and key network regulators (KNRs). From these analyses, KLF16 emerged as an important TF in osteogenic differentiation. We demonstrate that overexpression of Klf16 in vitro inhibited osteogenic differentiation and mineralization, while Klf16+/- mice exhibited increased bone mineral density, trabecular number, and cortical bone area. Our study underscores the complexity of osteogenic differentiation and identifies novel regulatory genes such as KLF16, which plays an inhibitory role in osteogenic differentiation both in vitro and in vivo.
Article activity feed
-
-
-
Author response
eLife Assessment
The authors investigated KLF Transcription Factor 16 (KLF16) as an inhibitor of osteogenic differentiation, which plays a critical role in bone development, metabolism and repair. The results of the study are valuable as they could help to facilitate future research on the regulation of osteogenesis in vitro and in vivo. However, the evidence overall is incomplete, as validation by knockout mouse models would help to strengthen the conclusions.
We appreciate the editors’ evaluation and recognition of the importance of our research. The primary goal and value of our study is to provide robust bioinformatics analyses of 20 independent iPSC lines, which can lead to the identification of novel genes involved in osteogenic differentiation. The identification of KLF16 serves to illustrate this goal. A …
Author response
eLife Assessment
The authors investigated KLF Transcription Factor 16 (KLF16) as an inhibitor of osteogenic differentiation, which plays a critical role in bone development, metabolism and repair. The results of the study are valuable as they could help to facilitate future research on the regulation of osteogenesis in vitro and in vivo. However, the evidence overall is incomplete, as validation by knockout mouse models would help to strengthen the conclusions.
We appreciate the editors’ evaluation and recognition of the importance of our research. The primary goal and value of our study is to provide robust bioinformatics analyses of 20 independent iPSC lines, which can lead to the identification of novel genes involved in osteogenic differentiation. The identification of KLF16 serves to illustrate this goal. A thorough analysis of the function of any single gene both in vitro and in vivo is beyond the initial scope of this study. To validate KLF16’s inhibitory role in osteogenic differentiation, we provided evidence showing overexpression of Klf16 suppressed osteogenic differentiation in vitro, and Klf16+/- mice exhibited enhanced bone mineral content and density in vivo.
Public Reviews:
Reviewer #1 (Public review):
Summary:
In this manuscript, Ru and colleagues investigated regulatory gene interactions during osteogenic differentiation. By profiling transcriptomic changes during mesenchymal stem cell differentiation, they identified KLF16 as a key transcription factor that inhibits osteogenic differentiation and mineralization. It was found that overexpression of KLF16 suppressed osteogenesis in vitro, while Klf16+/- mice exhibited enhanced bone density, underscoring its regulatory role in bone formation.
Strengths:
(1) Bioinformatics is strong and comprehensive.
(2) Identification of KLF16 in osteoblast differentiation is exciting and innovative.
We appreciate the reviewer’s comments on our bioinformatic analyses of MSC osteogenic differentiation and the identification of KLF16 as a new osteogenesis regulator. The differentiation of iPSC-derived MSCs to OBs serves as a valuable model for investigating gene expression and regulatory networks in osteogenic differentiation. This study provides insights into the complex and dynamic regulation of the transcriptomic landscape in osteogenic differentiation and supplies a foundational resource for additional investigation into normal bone formation and the mechanisms underlying pathological conditions.
Weaknesses:
(1) The mechanism of KLF16 function is not studied.
(2) Studies of KLF16 in bone development, from both in vitro and in vivo perspectives, are descriptive.
Our study aims to apply rigorous bioinformatic analyses of 20 iPSC lines to identify novel genes involved in osteogenic differentiation. With this strategy, we successfully identified KLF16 as a regulator of osteogenic differentiation. We validated this with both in vitro and in vivo models even though we had limited availability of Klf16 knockout mice when the study was conducted. We demonstrated that overexpression of Klf16 suppressed osteogenesis in vitro, while Klf16+/- mice exhibited increased bone mineral density, trabecular number, and cortical bone area, highlighting its role in bone formation. With these mice now available, further investigation into the mechanism of KLF16's function is possible.
(3) Findings in bioinformatics analysis are mostly redundant with previous studies in the field, and can be simplified.
We compared our bulk RNA-seq data with our previously published single-cell RNA-seq (scRNA-seq) data generated from iPSC-induced cells during osteogenic differentiation (Housman et al., 2022). The purpose is to corroborate the expression patterns of the genes we focused on during osteogenic differentiation. We found similar differential expression patterns in a pseudobulk analysis of the scRNA-seq data, even though there are significant differences between these two studies, including: cell culture conditions, sequencing approaches (bulk vs. single cell), goals of the studies (key TF drivers of osteoblast differentiation vs. mapping differentiation stages and inter-species gene programs in human and chimp), and findings (identification of TFs vs. identification of interspecific regulatory differences) .
Importantly, we performed network analyses to identify key transcription factors, which were not redundant with previous studies. We constructed a transcription factor regulatory network analysis during human osteogenic differentiation, and identified a network organized into five interactive modules. The most exciting finding was the identification of KLF16 as one of the strongest regulators in Module 5 (Figure 3), which previously was not demonstrated to be involved in bone formation. We also demonstrated known TF genes regulating osteogenic differentiation in these modules, and performed gene ontology (GO) and reactome pathway (RP) analyses for regulatory functions and pathways specific to each module. To clarify that our findings do not overlap with previous studies, we will revise the manuscript focusing on Module 5 and simplify the description of the bioinformatics analysis as the reviewer suggested.
Reviewer #2 (Public review):
In their manuscript with the title "Integrated transcriptomic analysis of human induced pluripotent stem cell (iPSC)-derived osteogenic differentiation reveals a regulatory role of KLF16", Ru et al. have analyzed the gene expression changes during the osteogenic differentiation of iPSC-derived mesenchymal stem/stromal cells into preosteoblasts and osteoblasts. As part of the computational analyses, they have investigated the transcription factor regulatory network mediating this differentiation process, which has also led to the identification of the transcription factor KLF16. Overexpression experiments in vitro and the analysis of heterozygous KLF16 knockout mice in vivo indicate that KLF16 is an inhibitor of osteogenic differentiation.
The integrated analysis of iPSC bulk transcriptomic data is a major strength of the study, and it is also great that the authors provide deeper functional characterization of the transcription factor KLF16, one of the newly identified candidate regulators of osteogenic differentiation.
We appreciate the reviewer’s summary and comments on the strength of our bioinformatic analyses of iPSC/MSC osteogenic differentiation and the deep functional characterization of the KLF16, as well as the novelty of our findings.
However, characterization of KLF16 expression in the mouse and validation of the knockout model are currently lacking. Alternative explanations for the mutant phenotype should be considered to improve the strength of the conclusions.
If all issues can be addressed, the study would provide an important resource for the field that would facilitate future research on the regulation of osteogenesis in vitro and in vivo, with potential implications for preclinical and clinical research as well as bioengineering.
We appreciate the reviewer’s valuable suggestions. Klf16 is highly expressed in mandibular, maxillary and tail mesenchyme at embryonic Day 12 (D'Souza et al., 2002), indicating its role in early bone development. We will further characterize the expression of Klf16 in mice, especially in the developing bones.
We identified Klf16 as a potential regulator of osteogenic differentiation, and then validated this with both in vitro and in vivo models. Overexpression of Klf16 suppressed osteogenesis in vitro, and Klf16+/- mice showed increased bone mineral content and density, indicating its regulatory role in bone formation. We agree with the reviewer that the bone phenotypes of Klf16 knockout mice potentially can be affected by other factors in addition to osteogenic differentiation. As both bone formation and resorption are critical for bone development, we evaluated osteoclastogenesis in the Klf16+/- mice by analyzing the expression of osteoclast marker CALCR and regulator RANKL in the femurs of the Klf16+/- mice. Neither CALCR nor RANKL decreased in the bone of Klf16+/- mice, indicating that osteoclastogenesis is not decreased; therefore, increased bone mineral content and density in the mutant mice is more likely attributed to enhanced bone formation rather than reduced resorption by osteoclasts. Additionally, we will discuss other alternative explanations for the bone phenotypes of Klf16 knockout mice as suggested by the reviewer.
References
D'Souza, U. M., Lammers, C.-H., Hwang, C. K., Yajima, S. and Mouradian, M. M. (2002). Developmental expression of the zinc finger transcription factor DRRF (dopamine receptor regulating factor). Mechanisms of Development 110, 197-201.
Housman, G., Briscoe, E. and Gilad, Y. (2022). Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model. PLOS Genetics 18, e1010073-e1010073.
-
eLife Assessment
The authors investigated KLF Transcription Factor 16 (KLF16) as an inhibitor of osteogenic differentiation, which plays a critical role in bone development, metabolism and repair. The results of the study are valuable as they could help to facilitate future research on the regulation of osteogenesis in vitro and in vivo. However, the evidence overall is incomplete, as validation by knockout mouse models would help to strengthen the conclusions.
-
Reviewer #1 (Public review):
Summary:
In this manuscript, Ru and colleagues investigated regulatory gene interactions during osteogenic differentiation. By profiling transcriptomic changes during mesenchymal stem cell differentiation, they identified KLF16 as a key transcription factor that inhibits osteogenic differentiation and mineralization. It was found that overexpression of KLF16 suppressed osteogenesis in vitro, while KLF16⁺/⁻ mice exhibited enhanced bone density, underscoring its regulatory role in bone formation.
Strengths:
(1) Bioinformatics is strong and comprehensive.
(2) Identification of KLF16 in osteoblast differentiation is exciting and innovative.
Weaknesses:
(1) The mechanism of KLF16 function is not studied.
(2) Studies of KLF16 in bone development, from both in vitro and in vivo perspectives, are descriptive.
(3) …
Reviewer #1 (Public review):
Summary:
In this manuscript, Ru and colleagues investigated regulatory gene interactions during osteogenic differentiation. By profiling transcriptomic changes during mesenchymal stem cell differentiation, they identified KLF16 as a key transcription factor that inhibits osteogenic differentiation and mineralization. It was found that overexpression of KLF16 suppressed osteogenesis in vitro, while KLF16⁺/⁻ mice exhibited enhanced bone density, underscoring its regulatory role in bone formation.
Strengths:
(1) Bioinformatics is strong and comprehensive.
(2) Identification of KLF16 in osteoblast differentiation is exciting and innovative.
Weaknesses:
(1) The mechanism of KLF16 function is not studied.
(2) Studies of KLF16 in bone development, from both in vitro and in vivo perspectives, are descriptive.
(3) Findings in bioinformatics analysis are mostly redundant with previous studies in the field, and can be simplified.
-
Reviewer #2 (Public review):
In their manuscript with the title "Integrated transcriptomic analysis of human induced pluripotent stem cell (iPSC)-derived osteogenic differentiation reveals a regulatory role of KLF16", Ru et al. have analyzed the gene expression changes during the osteogenic differentiation of iPSC-derived mesenchymal stem/stromal cells into preosteoblasts and osteoblasts. As part of the computational analyses, they have investigated the transcription factor regulatory network mediating this differentiation process, which has also led to the identification of the transcription factor KLF16. Overexpression experiments in vitro and the analysis of heterozygous KLF16 knockout mice in vivo indicate that KLF16 is an inhibitor of osteogenic differentiation.
The integrated analysis of iPSC bulk transcriptomic data is a major …
Reviewer #2 (Public review):
In their manuscript with the title "Integrated transcriptomic analysis of human induced pluripotent stem cell (iPSC)-derived osteogenic differentiation reveals a regulatory role of KLF16", Ru et al. have analyzed the gene expression changes during the osteogenic differentiation of iPSC-derived mesenchymal stem/stromal cells into preosteoblasts and osteoblasts. As part of the computational analyses, they have investigated the transcription factor regulatory network mediating this differentiation process, which has also led to the identification of the transcription factor KLF16. Overexpression experiments in vitro and the analysis of heterozygous KLF16 knockout mice in vivo indicate that KLF16 is an inhibitor of osteogenic differentiation.
The integrated analysis of iPSC bulk transcriptomic data is a major strength of the study, and it is also great that the authors provide deeper functional characterization of the transcription factor KLF16, one of the newly identified candidate regulators of osteogenic differentiation.
However, characterization of KLF16 expression in the mouse and validation of the knockout model are currently lacking. Alternative explanations for the mutant phenotype should be considered to improve the strength of the conclusions.
If all issues can be addressed, the study would provide an important resource for the field that would facilitate future research on the regulation of osteogenesis in vitro and in vivo, with potential implications for preclinical and clinical research as well as bioengineering.
-
-