Smooth muscle protein 22α‐Cre recombination in resting cardiac fibroblasts and hematopoietic precursors

概要

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Smooth muscle protein 22α‑Cre
recombination in resting cardiac
fibroblasts and hematopoietic
precursors
Shinya Ikeda1*, Sachiko Sugioka1, Takeshi Kimura1,2 & Noboru Ashida1*
The Cre-loxP system has been widely used for cell- or organ-specific gene manipulation, but it is
important to precisely understand what kind of cells the recombination takes place in. Smooth muscle
22α (SM22α)-Cre mice have been utilized to alter genes in vascular smooth muscle cells (VSMCs),
activated fibroblasts or cardiomyocytes (CMs). Moreover, previous reports indicated that SM22α-Cre
is expressed in adipocytes, platelets or myeloid cells. However, there have been no report of whether
SM22α-Cre recombination takes place in nonCMs in hearts. Thus, we used the double-fluorescent Cre
reporter mouse in which GFP is expressed when recombination occurs. Immunofluorescence analysis
demonstrated that recombination occurred in resting cardiac fibroblasts (CFs) or macrophages, as
well as VSMCs and CMs. Flow cytometry showed that some CFs, resident macrophages, neutrophils,
T cells, and B cells were positive for GFP. These results prompted us to analyze bone marrow cells,
and we observed GFP-positive hematopoietic precursor cells (HPCs). Taken together, these results
indicated that SM22α-Cre-mediated recombination occurs in resting CFs and hematopoietic cell
lineages, including HPCs, which is a cautionary point when using SM22α-Cre mice.
テキスト

テキスト

The Cre-loxP system enables us to edit genes of interest in a tissue- or cell-specific manner. However, it goes
without saying that researchers should understand precisely which kind of cells are subjected to recombination.
In cardiovascular research, Smooth muscle 22α (SM22α)-Cre has been widely used to manipulate genes in vascular smooth muscle cells (VSMCs), activated fibroblasts and cardiomyocytes (CMs)1–4. Moreover, SM22α-Cre
has been reported to be expressed in adipocytes, platelets and multiple lineages of myeloid c­ ells5,6. However,
there has been no report of whether SM22α-Cre recombination takes place in nonCMs in hearts. We designed
a study to answer this question.
In this study, we used double-fluorescent Cre reporter ­mice7 mated with SM22α-Cre (SMmTmG) mice and
analyzed relevant indices by immunofluorescence and flow cytometry.

Result

SM22α‑Cre is expressed in quiescent cardiac fibroblasts (CFs) and macrophages in adult and
embryonic hearts.  We evaluated in which types of cells SM22α-Cre recombination takes place in adult

and embryonic murine steady-state hearts. Similar to a previous ­study1, VSMCs and approximately 90% of CMs
expressed GFP (Fig. 1a,b). Surprisingly, there were many GFP and Vimentin positive cells even though there
were no myofibroblasts in murine steady-state ­hearts8 (Fig. 1b,c). This finding suggested that SM22α-Cre was
expressed in resting CFs. Moreover, GFP was detected in some macrophages (CD68+) (Fig. 1c). However, there
were no GFP-positive endothelial cells (CD31+) (Fig. 1c), which are the most numerous cell type in the murine
heart.

Flow cytometric analysis of SM22α‑Cre expression in hearts.  We were interested in the precise GFP

percentages among specific cell types, such as fibroblasts and macrophages. Flow cytometric analysis of adult
murine hearts revealed that fibroblasts (CD31−, CD45−, PDGFRa+) (65.6 ± 3.5%) had a GFP-positive group,
while very few endothelial cells (CD31+ and CD45−) (1.3 ± 0.2%) expressed GFP, which was in agreement with
the immunofluorescent analysis (Fig. 2a–c). Detailed investigation of the immune cells revealed that some GFP-

1

Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara‑cho,
Shogoin, Sakyo‑ku, Kyoto  606‑8507, Japan. 2Hirakata Kohsai Hospital, Osaka, Japan. *email: shinya_i210@
kuhp.kyoto-u.ac.jp; nashida@kuhp.kyoto-u.ac.jp

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Figure 1.  Immunofluorescence analysis of the hearts of SMmTmG mice. (a) A representative image of the
whole heart without any staining. Approximately 90% of CMs expressed GFP. Original magnification:  ×5.
(b) The expression of αSMA in cardiac vessels and the myocardium. VSMCs expressed GFP. Original
magnification:  ×63. Scale bars: 40 μm. (c) The expression of the cell-specific markers, GFP and dTomato (left
panels) and details (right 4 panels) in adult and embryonic hearts. There were some GFP+ CFs (Vimentin+) and
GFP+ macrophages (CD68+), but no GFP+ endothelial cells (CD31+). Original magnification: × 63. Scale bars:
40 μm (original images) or 10 μm (details).

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Figure 2.  Flow cytometric analysis of nonCMs in SMmTmG mice. (a) The representative gating schema of
murine hearts. (b) GFP expression in each cell type in the heart. (c) Summary of GFP expression in the heart
(fibroblasts and endothelial cells: n = 5, others: n = 3).

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positive populations were present among macrophages (CD45+, CD11b+, CD64+, Ly6c low) (18.9 ± 0.8%) and
neutrophils (CD45+, CD11b+, Ly6G+) (27.7 ± 2.9%) as described in a previous ­paper5. Furthermore, some cardiac resident macrophages (CD45+, CD11b+, CD64+, Ly6c low, CCR2−, MHC2 low) (18.4 ± 1.1%) and MHC2
high macrophage (CD45+, CD11b+, CD64+, Ly6c low, CCR2-, MHC2 high) (20.0 ± 1.0%), both of which are
derived from the yolk sac or fetal l­iver9,10, expressed GFP (Fig.  2a–c). Surprisingly, among T cells (CD45+,
CD11b−, CD3ε+) (26.8 ± 3.6%) and B cells (CD45+, CD11b−, B220+) (25.8 ± 3.6%), there were GFP-positive
populations, unlike in a previous ­paper5 (Fig. 2a–c).

Flow cytometric analysis of SM22α‑Cre expression in peripheral blood and bone marrow.  We
assessed whether SM22α-Cre was expressed in adult murine peripheral blood and bone marrow because we
identified GFP-positive groups among T cells and B cells in the heart. Consistent with the results obtained
in the heart, GFP was detected in monocytes (27.9 ± 1.0%), neutrophils (28.2 ± 2.3%), T cells (32 ± 3.4%) and
B cells (27.8 ± 2.7%) in peripheral blood (Fig.  3a–c). Surprisingly, phenotypically defined putative long-term
hematopoietic stem cells (LT-HSCs, Lineage−, c-Kit+, Sca1+, CD150+, CD48−) (23.5 ± 0.8%) and short-term
HSCs (ST-HSCs, Lineage-, c-Kit+, Sca1+, CD150−, CD48−) (26.8 ± 1.4%), multipotent progenitors (MPPs, Lineage−, c-Kit+, Sca1+, CD150-, CD48 +) (31.9 ± 2.3%)11, downstream myeloid lineages, such as common myeloid
progenitors (CMPs, Lineage−, c-Kit+, Sca1−, CD34+, CD16/32−) (23.8 ± 2.7%), granulocyte macrophage progenitors (GMPs, Lineage-, Sca-1−, c-Kit+, CD34+, CD16/32+, CD115−) (25.3 ± 2.9%), monocyte-macrophage
dendritic cell progenitors (MDPs, Lineage−, Sca-1−, c-Kit+, CD34+, CD16/32+, CD115+) (26.0 ± 4.0%), and
megakaryocyte-erythrocyte progenitors (MEPs, Lineage-, Sca-1−, c-Kit+, CD34−, CD16/32−) (23.1 ± 1.6%)12,
and the lymphoid lineages, including common lymphoid progenitors (CLPs, Lineage-, Sca-1 low, c-Kit low,
CD135+, CD127+) (22.1 ± 2.4%), innate lymphoid cells (ILCs, Lineage−, Sca-1+, c-Kit−) (26.4 ± 2.3%)13 had
GFP-positive populations (Fig. 3d,e and Supplementary Fig. 1).
In vitro, SM22α‑Cre was expressed in CFs, but not in bone marrow‑derived macrophages
(BMDMs).  We investigated whether cells isolated from SM22α-Cre mice were useful for editing genes in vitro

because the efficiency of gene editing, such as that mediated by a lentivirus, still has room for consideration.
Almost all isolated CFs (90.3 ± 0.5%) can become GFP + cells by being seeded on a culture dish because
fibroblasts become myofibroblasts on a culture ­dish14 (Fig. 4a–c). However, TGF-β stimulation can upregulate
Tagln (the SM22α gene) and Acta2 (the αSMA gene) expression in BMDMs, but the expression levels were so
much lower than those in fibroblasts that SM22α-Cre recombination could not be used in BMDMs (Fig. 4d,e
and Supplementary Fig. 2).
Furthermore, we analyzed whether differentiation facilitates SM22α-Cre recombination. Flow cytometric
analysis showed that differentiation had no effect on recombination under TGF-β stimulation (Fig. 4f,g).

Discussion

SM22α-Cre recombination has not been fully assessed in murine hearts, although SM22α-Cre has been widely
used to alter VSMCs, activated fibroblasts and CMs in cardiovascular r­ esearch2–4. This study offers further insights
into the target of SM22α-Cre and how to lead recombination in some cells. First, SM22α-Cre recombination
occurs in 65% of resting CFs. Second, SM22α-Cre recombination occurs in cardiac T and B cells, in addition to
myeloid cells. Third, recombination also takes place in putative HSCs. Finally, CFs have the capacity to express
SM22α-Cre. However, TGF-β, which is well known to induce ­Tagln15–17, cannot induce SM22α-Cre recombination in BMDMs.
One important finding in the present study is that there are some GFP-positive populations among resting
CFs from embryos. SM22α is a well-known marker of activated ­fibroblasts18. In vivo, SM22α-Cre cannot be used
to target only activated CFs, but could be used to manage quiescent CFs. As stated in previous p
­ apers5,6,19, we
recommend other Cre recombination systems, such as Postn-Cre when focusing on activated CFs.
Next, SM22α-Cre is expressed in some immune cells, including phenotypically defined putative HSCs. It
was unlike the results of a previous ­paper5, but Zovein et al. observed SM22α-Cre recombination at E11.5 in
the aortic-gonado-mesonephros (AGM) region, the origin of H
­ SCs20. This is likely to support the results on the
recombination in HSCs, but further in vivo repopulating experiments would confirm this conclusion. ...

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