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. 2020 Jan 11;9(1):191.
doi: 10.3390/cells9010191.

Cytokine Directed Chondroblast Trans-Differentiation: JAK Inhibition Facilitates Direct Reprogramming of Fibroblasts to Chondroblasts

Affiliations

Cytokine Directed Chondroblast Trans-Differentiation: JAK Inhibition Facilitates Direct Reprogramming of Fibroblasts to Chondroblasts

Perla Cota et al. Cells. .

Abstract

Osteoarthritis (OA) is a degenerative disease of the hyaline articular cartilage. This disease is progressive and may lead to disability. Researchers proposed many regenerative approaches to treat osteoarthritis, including stem cells. Trans-differentiation of a fully differentiated cell state directly into another different differentiated cell state avoids the disadvantages of fully reprogramming cells to induced pluripotent stem cells (iPSCs) in terms of faster reprogramming of the needed cells. Trans-differentiation also reduces the risk of tumor formation by avoiding the iPSC state. OSKM factors (Oct4, Sox2, Klf4, and cMyc) accompanied by the JAK-STAT pathway inhibition, followed by the introduction of specific differentiation factors, directly reprogrammed mouse embryonic fibroblasts to chondroblasts. Our results showed the absence of intermediate induced pluripotent stem cell formation. The resulting aggregates showed clear hyaline and hypertrophic cartilage. Tumor formation was absent in sub-cutaneous capsules transplanted in SCID mice.

Keywords: cartilage regeneration; cellular reprogramming; osteoarthritis; trans-differentiation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Transduction efficiency of MEFs using lentiviral vectors. To evaluate the efficiency of transduction, we utilized the fluorescence reporter gene Ds-Red and transduced MEFs as outlined in the materials and methods. (a) Transduced MEFs are fluorescent red under the microscope. Scale bar 50 μm. (b,c) Transgene expression reached a maximum on day 7 and maintained the signal up to day 9. (d,e) Upregulation of pluripotency gene expression in the presence of iPSC-inductive media (+LIF/−JI1), and trans-differentiation media (−LIF/+JI1). Nanog and Rex-1 are significantly upregulated 3-fold and 10-fold, respectively, in cells under iPSC reprogramming media. **** p < 0.0001.
Figure 2
Figure 2
Exogenous, but not endogenous Oct4 expression during the first stage of trans- differentiation. The figure shows (a) the total signal of Oct4 (exogenous and endogenous) at every time point, and (b) no endogenous Oct4 at any time point. (c) Exogenous Oct4 signal was present since day 2 with the highest signal shown at day 9 indicating that partial reprogramming is occurring. (d) Histogram demonstrating the log change in exogenous Oct4 compared to the absence of log change in endogenous Oct4. The absence of log change on endogenous Oct4 and the log change on exogenous Oct4 demonstrated the absence of iPSCs formation.
Figure 3
Figure 3
A negligible number of chondroblasts present in the starting MEFs population. 2-D histogram plotted with fluorescent intensity from fibroblast (MEFs) marked with H2kk (APC) and Col2 (FITC) antibodies analyzed by flow cytometry. (a) The horizontal-vertical lines divide the cells into four quadrants, which represent cells that were not marked or did not have an expression of those proteins (bottom left), cells with H2kk expression (top left), cells with Col2 expression (bottom right) and cells with expression of both proteins (top right). (b) In total, 96.6% of the MEFs population stained positive for H2kk, whereas 0.8% positive for both H2kk and Col2. (c) Expression of Sox9 in MEFs cultured with chondrogenic media during different time points is comparable to control MEFs. (dg) Chondroblast gene expression is different in transdifferentiated cells compared to MEFs. OSKM-transduced MEFs were transdifferentiated (−LIF+JI1) and then analyzed for chondrogenic gene expression (Sox9, Col2, Col10, and Mmp13) with a comparison to MEFs (starting cell population). Gene expression data normalized to MEFs. The figure shows that the expression of Sox9 was five times higher in transdifferentiated cells than in MEFs, with a significant upregulation. * p < 0.050.
Figure 4
Figure 4
Effect of JI1 to the chondrogenic gene expression kinetics of transdifferentiated aggregates. Cells were transdifferentiated (day 2 to 36) under a conducive and non-conduce media. (a) Sox9 expression demonstrated its upregulation on day 12 (2nd stage) with later gene expression reduction toward the end of the process. Levels of expression were similar for both conditions. (b) Col2 expression was not upregulated in either condition at any time in the process. (c) Col10 was expressed at a higher level in cells under conducive media and expression levels increased with time. No Col10 expression under non-conducive media. (d) The Mmp13 gene was upregulated under conducive media on day 15, and this continued up to day 36. The Mmp13 gene was not present in non-conducive media. (e,f) MEFs do not undergo spontaneous chondrogenesis during the first stage of trans-differentiation either in the presence or absence of LIF or JI1. OSKM-transduced MEFs were cultured in three different conditions (−LIF/+JI1, −LIF/−JI1 and +LIFJI1) and samples on days 2, 6, and 9 were analyzed for Sox9 and Col2 gene expression. Both Sox9 and Col2 genes showed insignificant upregulation at all time points and conditions.
Figure 5
Figure 5
Bone markers gene expression is stimulated in the absence of JI1 during trans-differentiation. (a,b) Downregulation of Runx2 and Sp7 in the presence of JI1. (c) The marker ACP5 showed the highest expression (7-fold) in non-conducive media compared to the conducive conditions. ** p < 0.010.
Figure 6
Figure 6
Cartilage structure and glycosaminoglycan (GAG) are present in the transdifferentiated aggregates. (a) Compact cartilage aggregates in culture. (b) Cartilage-like tissue stained by H & E. (c) Hyaline cartilage-like tissue stained by H & E with sparse chondroblasts in the lacunae structure (Scale bar 10 μm). (d) (GAG) deposition stained by Safranin-O. (e) (GAG) deposition stained by Alcian blue. (f) H & E staining of hyaline chondroblasts and hyaline matrix found in vivo following transplantation in mice. (g,h) H & E staining of hypertrophic chondroblasts in a subcutaneous capsule. (i) H & E staining of developing limb bud in a mouse embryo showing natural, normal hyaline (right arrow) and hypertrophic (left arrow) chondroblasts (Scale bar 25μm). (j) H & E staining of chondroblast aggregates differentiated from mouse iPSCs showing hyaline cartilage-like tissue with chondroblasts in lacunae (left arrow) and hypertrophic chondroblasts (right arrow) (Scale bar 10 μm). (k) IPSCs derived teratoma showing heterogenous tissue formation (Scale bar 25 μm).
Figure 7
Figure 7
Schematic of the methodology used in this study. Retroviral transduction partially reprogramed MEFs in the absence of LIF and the addition of JI1. This approach put the cells into a plastic state where the chromatin is open enough for the chemicals and small molecules in the chondrogenic media to reprogram cells to chondroblasts without reaching the iPSCs state directly.
Figure 8
Figure 8
Schematics of the LIF/JAK-STAT3 pathway active and blocked by a JAK inhibitor. (a) LIF binds to its Gp130/LIFR-β and activates the JAK-STAT3 pathway. Its activation leads to the direct stimulation of the pluripotency circuitry transcription factors in the nucleus for pluripotency reprogramming. (b) When JAK-STAT and no other pathway is inactivated, the stimulation of the pluripotency circuitry decays, and the reprogramming process is affected.

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