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. 2020 Jan;14(1):29-44.
doi: 10.1002/term.2961. Epub 2019 Dec 18.

Transcriptome dynamics of long noncoding RNAs and transcription factors demarcate human neonatal, adult, and human mesenchymal stem cell-derived engineered cartilage

Daniel J Vail  1 , Rodrigo A Somoza  2 , Arnold I Caplan  2 , Ahmad M Khalil  1   3
Affiliations

Transcriptome dynamics of long noncoding RNAs and transcription factors demarcate human neonatal, adult, and human mesenchymal stem cell-derived engineered cartilage

Daniel J Vail et al. J Tissue Eng Regen Med. 2020 Jan.

Abstract

The engineering of a native-like articular cartilage (AC) is a long-standing objective that could serve the clinical needs of millions of patients suffering from osteoarthritis and cartilage injury. An incomplete understanding of the developmental stages of AC has contributed to limited success in this endeavor. Using next generation RNA sequencing, we have transcriptionally characterized two critical stages of AC development in humans-that is, immature neonatal and mature adult, as well as tissue-engineered cartilage derived from culture expanded human mesenchymal stem cells. We identified key transcription factors (TFs) and long noncoding RNAs (lncRNAs) as candidate drivers of the distinct phenotypes of these tissues. AGTR2, SCGB3A1, TFCP2L1, RORC, and TBX4 stand out as key TFs, whose expression may be capable of reprogramming engineered cartilage into a more expandable and neonatal-like cartilage primed for maturation into biomechanically competent cartilage. We also identified that the transcriptional profiles of many annotated but poorly studied lncRNAs were dramatically different between these cartilages, indicating that lncRNAs may also be playing significant roles in cartilage biology. Key neonatal-specific lncRNAs identified include AC092818.1, AC099560.1, and KC877982. Collectively, our results suggest that tissue-engineered cartilage can be optimized for future clinical applications by the specific expression of TFs and lncRNAs.

Keywords: cartilage; hMSCs; long noncoding RNAs; tissue engineering; transcription factors.

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Conflict of interest statement

Conflict of interest:

The authors declare that there is no conflict of interest to be declared.

Figures

Figure 1.
Figure 1.. Gene expression comparison of human adult vs. neonatal cartilage.
A. Venn diagrams comparing all transcripts (left), TFs (middle), and lincRNAs (right) consistently expressed (all replicates TPM ≥ 1) in adult and neonatal AC. B. Heat map of all differentially expressed genes between adult and neonatal AC samples with hierarchical clustering of biological replicate groupings. C. Two dimensional principal component analysis comparing likeness of adult and neonatal AC by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. PANTHER GO pathway analysis demonstrating the pathways overrepresented by differentially expressed genes. E. Expression patterns of potential molecular driver TFs for neonatal AC as defined by a minimum expression of 1 TPM in each neonatal replicate and ≤ TPM of 1 in all adult replicates. F. Expression patterns of potential molecular driver TFs for adult AC as defined by a minimum expression of 1 TPM in each adult replicate and ≤ TPM of 1 in all neonatal replicates.
Figure 1.
Figure 1.. Gene expression comparison of human adult vs. neonatal cartilage.
A. Venn diagrams comparing all transcripts (left), TFs (middle), and lincRNAs (right) consistently expressed (all replicates TPM ≥ 1) in adult and neonatal AC. B. Heat map of all differentially expressed genes between adult and neonatal AC samples with hierarchical clustering of biological replicate groupings. C. Two dimensional principal component analysis comparing likeness of adult and neonatal AC by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. PANTHER GO pathway analysis demonstrating the pathways overrepresented by differentially expressed genes. E. Expression patterns of potential molecular driver TFs for neonatal AC as defined by a minimum expression of 1 TPM in each neonatal replicate and ≤ TPM of 1 in all adult replicates. F. Expression patterns of potential molecular driver TFs for adult AC as defined by a minimum expression of 1 TPM in each adult replicate and ≤ TPM of 1 in all neonatal replicates.
Figure 1.
Figure 1.. Gene expression comparison of human adult vs. neonatal cartilage.
A. Venn diagrams comparing all transcripts (left), TFs (middle), and lincRNAs (right) consistently expressed (all replicates TPM ≥ 1) in adult and neonatal AC. B. Heat map of all differentially expressed genes between adult and neonatal AC samples with hierarchical clustering of biological replicate groupings. C. Two dimensional principal component analysis comparing likeness of adult and neonatal AC by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. PANTHER GO pathway analysis demonstrating the pathways overrepresented by differentially expressed genes. E. Expression patterns of potential molecular driver TFs for neonatal AC as defined by a minimum expression of 1 TPM in each neonatal replicate and ≤ TPM of 1 in all adult replicates. F. Expression patterns of potential molecular driver TFs for adult AC as defined by a minimum expression of 1 TPM in each adult replicate and ≤ TPM of 1 in all neonatal replicates.
Figure 1.
Figure 1.. Gene expression comparison of human adult vs. neonatal cartilage.
A. Venn diagrams comparing all transcripts (left), TFs (middle), and lincRNAs (right) consistently expressed (all replicates TPM ≥ 1) in adult and neonatal AC. B. Heat map of all differentially expressed genes between adult and neonatal AC samples with hierarchical clustering of biological replicate groupings. C. Two dimensional principal component analysis comparing likeness of adult and neonatal AC by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. PANTHER GO pathway analysis demonstrating the pathways overrepresented by differentially expressed genes. E. Expression patterns of potential molecular driver TFs for neonatal AC as defined by a minimum expression of 1 TPM in each neonatal replicate and ≤ TPM of 1 in all adult replicates. F. Expression patterns of potential molecular driver TFs for adult AC as defined by a minimum expression of 1 TPM in each adult replicate and ≤ TPM of 1 in all neonatal replicates.
Figure 1.
Figure 1.. Gene expression comparison of human adult vs. neonatal cartilage.
A. Venn diagrams comparing all transcripts (left), TFs (middle), and lincRNAs (right) consistently expressed (all replicates TPM ≥ 1) in adult and neonatal AC. B. Heat map of all differentially expressed genes between adult and neonatal AC samples with hierarchical clustering of biological replicate groupings. C. Two dimensional principal component analysis comparing likeness of adult and neonatal AC by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. PANTHER GO pathway analysis demonstrating the pathways overrepresented by differentially expressed genes. E. Expression patterns of potential molecular driver TFs for neonatal AC as defined by a minimum expression of 1 TPM in each neonatal replicate and ≤ TPM of 1 in all adult replicates. F. Expression patterns of potential molecular driver TFs for adult AC as defined by a minimum expression of 1 TPM in each adult replicate and ≤ TPM of 1 in all neonatal replicates.
Figure 1.
Figure 1.. Gene expression comparison of human adult vs. neonatal cartilage.
A. Venn diagrams comparing all transcripts (left), TFs (middle), and lincRNAs (right) consistently expressed (all replicates TPM ≥ 1) in adult and neonatal AC. B. Heat map of all differentially expressed genes between adult and neonatal AC samples with hierarchical clustering of biological replicate groupings. C. Two dimensional principal component analysis comparing likeness of adult and neonatal AC by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. PANTHER GO pathway analysis demonstrating the pathways overrepresented by differentially expressed genes. E. Expression patterns of potential molecular driver TFs for neonatal AC as defined by a minimum expression of 1 TPM in each neonatal replicate and ≤ TPM of 1 in all adult replicates. F. Expression patterns of potential molecular driver TFs for adult AC as defined by a minimum expression of 1 TPM in each adult replicate and ≤ TPM of 1 in all neonatal replicates.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 2.
Figure 2.. Dynamic changes in gene expression are observed during hMSC differentiation in AC in vitro
A. Venn diagram of all transcripts, TFs, and lncRNAs for hMSCs versus all transcripts, TFs, and lncRNAs expressed at any point between pellet culture day 3 and day 21. B. Count of DEGs between consecutive days throughout pellet culture (DEGs defined here as any replicate TPM ≥ 1 and adjusted p-value ≤ 0.05). C. PCA comparing hMSCs and chondrogenic pellets at various time points throughout differentiation by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. D. Highlight of rapidly and highly expressed TFs (red) and transiently expressed TFs (blue). Also shows all other TFs consistently (mean TPM≥1) expressed throughout chondrogenesis not highlighted in (E) or (F) (gray). E. TFs increasing by four-fold between day 3 and day 21 of pellet culture. F. TFs decreasing by four-fold between day 3 and day 21 of pellet culture.. G. lncRNAs increasing by four-fold between day 3 and day 21 of pellet culture. H. lncRNAs decreasing by four-fold between day 3 and day 21 of pellet culture.
Figure 3.
Figure 3.. Human native cartilage show substantial gene expression differences with hMSC-derived cartilage in vitro
A. Venn diagram of day 21 pellets vs adult and neonatal AC (All, TFs, and lncRNAs). B. PCA comparing pellets and primary ACs by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. C. Heat map of DEGs for day 21 pellets vs neonatal and adult AC.
Figure 3.
Figure 3.. Human native cartilage show substantial gene expression differences with hMSC-derived cartilage in vitro
A. Venn diagram of day 21 pellets vs adult and neonatal AC (All, TFs, and lncRNAs). B. PCA comparing pellets and primary ACs by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. C. Heat map of DEGs for day 21 pellets vs neonatal and adult AC.
Figure 3.
Figure 3.. Human native cartilage show substantial gene expression differences with hMSC-derived cartilage in vitro
A. Venn diagram of day 21 pellets vs adult and neonatal AC (All, TFs, and lncRNAs). B. PCA comparing pellets and primary ACs by transcriptional profile of transcripts with at least one sample expressing at ≥ TPM of 1. C. Heat map of DEGs for day 21 pellets vs neonatal and adult AC.
Figure 4.
Figure 4.. Key Transcription factors and long non-coding RNAs that are either unique or shared between neonatal vs. adult cartilage and absent in in vitro chondrogenesis
A. TFs expressed (TPM ≥ 1) in primary tissues but never in hMSC chondrogenic differentiation (TPM < 1 days 3–21). B. lncRNAs expressed (TPM ≥ 5) in primary tissues but never in hMSC chondrogenic differentiation (TPM < 1 days 3–21).
Figure 4.
Figure 4.. Key Transcription factors and long non-coding RNAs that are either unique or shared between neonatal vs. adult cartilage and absent in in vitro chondrogenesis
A. TFs expressed (TPM ≥ 1) in primary tissues but never in hMSC chondrogenic differentiation (TPM < 1 days 3–21). B. lncRNAs expressed (TPM ≥ 5) in primary tissues but never in hMSC chondrogenic differentiation (TPM < 1 days 3–21).
Figure 5.
Figure 5.. Uniquely expressed lncRNAs in either neonatal or adult cartilage and example co-expression of lncRNAs with genomic co-occupant cartilage-related protein-coding gene
A. lncRNAs consistently expressed under stringent criteria (TPM≥5) in each primary AC but not in the other (TPM <1). B. Co-expression of COL15A1 with AL136084.3. C. Co-expression of ID2 with ID2-AS1.
Figure 5.
Figure 5.. Uniquely expressed lncRNAs in either neonatal or adult cartilage and example co-expression of lncRNAs with genomic co-occupant cartilage-related protein-coding gene
A. lncRNAs consistently expressed under stringent criteria (TPM≥5) in each primary AC but not in the other (TPM <1). B. Co-expression of COL15A1 with AL136084.3. C. Co-expression of ID2 with ID2-AS1.
Figure 5.
Figure 5.. Uniquely expressed lncRNAs in either neonatal or adult cartilage and example co-expression of lncRNAs with genomic co-occupant cartilage-related protein-coding gene
A. lncRNAs consistently expressed under stringent criteria (TPM≥5) in each primary AC but not in the other (TPM <1). B. Co-expression of COL15A1 with AL136084.3. C. Co-expression of ID2 with ID2-AS1.
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