Blastocyst complementation-based rat-derived heart generation reveals cardiac anomaly barriers to interspecies chimera development

Abstract

The use of pluripotent stem cells (PSCs) to generate functional organs via blastocyst complementation is a cutting-edge strategy in regenerative medicine. However, existing models that use this method for heart generation do not meet expectations owing to the complexity of heart development. Here, we investigated a Mesp1/2 deficient mouse model, which is characterized by abnormalities in the cardiac mesodermal cells. The injection of either mouse or rat PSCs into Mesp1/2 deficient mouse blastocysts led to successful heart generation. In chimeras, the resulting hearts were predominantly composed of rat cells; however, their functionality was limited to the embryonic developmental stage on day 12.5. These results present the functional limitation of the xenogeneic heart, which poses a significant challenge to the development in mouse-rat chimeras.

Keywords: Health sciences; biological sciences; cardiovascular medicine.

Graphical abstract

Figure 1

Analysis of the Mesp1-KO model in the heart with reverse blastocyst complementation (rBC) method (A) Schematic of reverse-blastocyst complementation (rBC) method. Mesp1-knockout (KO) embryonic stem cells (ESCs) expressing red fluorescent protein (RFP) were injected into the wild type (WT) embryo. Mesp1-KO ESCs+ WT chimeras were dissected to assess the contribution of the progeny of exogenous donor ESCs to heart tissue. (B) Strategy for generation of Mesp1-KO model. Both gRNA1 and gRNA2 were used to remove exon1 and exon 2 of the Mesp1 gene. Fw1-Rv1 primer set was used to detect the deletion of the Mesp1 gene. Fw2-Rv2 primer set was used to detect the WT allele of the Mesp1 gene. (C) Genotype of Mesp1-KO, Mesp1 heterozygous (Het), and Mesp1 WT ESCs. (D) Representative embryo and organs derived from RFP-expressing Mesp1-KO ESCs at E14.5. (T; Tail, K: Kidney, G: Gonad, B: Bladder, H: Heart, Li: Liver, Lu: Lung, S: Stomach, I: Intestine) Scale bars, 1 mm.

Figure 2

Analysis of Mesp1/2-DKO model using rBC method at E8.5 (A) Schematic of reverse-blastocyst complementation (rBC) method. Mesp1/2-double knockout (DKO) embryonic stem cells (ESCs) expressing red fluorescent protein (RFP) were injected into the wild type (WT) embryo. Mesp1/2-DKO ESCs+WT chimeras were dissected to assess the contribution of the progeny of exogenous donor ESCs to heart tissue. (B) Strategy for the generation of the Mesp1/2-DKO model. Both gRNA2 and gRNA3 were used to remove Mesp1 and Mesp2 genes. Fw1-Rv1 primer set was used to detect deletion of both Mesp1 and Mesp2 genes. Fw2-Rv2 primer set was used to detect no deletion of Mesp1 and Mesp2 genes. (C) Genotype of Mesp1/2-DKO and WT ESCs. (D) Representative images of embryos derived from RFP-expressing Mesp1/2-DKO ESCs at E8.5. Mesp1/2-DKO cells with higher chimerism showed abnormal shape. Scale bars, 1 mm. (E) Representative image of embryos derived from RFP-expressing Mesp1/2-DKO and WT ESCs at E8.5. RFP+ cells are not detected in the Mesp1/2-DKO chimera heart. Scale bars, 1 mm.

Figure 3

Analysis of Mesp1/2-DKO model using rBC method at E14.5 (A) Representative image of embryos and organs derived from red fluorescent protein (RFP)-expressing Mesp1/2-double knock out (DKO) ESCs at E14.5. (T; Tail, K: Kidney, G: Gonad, B: Bladder, H: Heart, Li: Liver, Lu: Lung, S: Stomach, I: Intestine) Scale bars, 1 mm. (B) Representative immunostaining image of cTnt and CD31 in the heart of Mesp1/2-DKO ESCs+WT or WT ESCs+WT chimera. Scale bars, 500 μm. (C) Flow cytometry analysis of tissues from the Mesp1/2-DKO ESCs+WT or WT ESCs+WT chimeras. The fold change shows that the chimerism of each tissue (heart, intestine, kidney, stomach, and tail) was divided into the chimerism of the lung in the chimeras. All values are expressed as mean ± standard deviation from at least triplicate experiments (n = 16 in Mesp1/2-DKO chimera; n = 9 in WT chimera, ∗∗∗: p < 0.01).

Figure 4

Analysis of the Mesp1/2-DKO model using the rBC method at 8 weeks (A) Representative image of chimera mice derived from Mesp1/2-double knock out (DKO) ESCs and WT cells or WT ESCs and WT cells at 8 weeks old. Mesp1/2-DKO ESCs+WT chimeras show clear shortened tail morphology (arrow). Scale bars, 1 cm. (B) Representative image of Alcian blue and Alizarin red staining of the tail from Mesp1/2-DKO ESCs+WT or WT ESC+WT chimeras. Scale bars, 1 cm. (C) Macroscopic images of the heart, kidney, and lung of chimera mice from red fluorescent protein (RFP) expressing Mesp1/2-DKO ESCs+WT or RFP expressing WT ESCs+WT. (n = 4 chimeras from Mesp1/2-DKO ESC or WT ESC injection) Scale bars, 1 cm.

Figure 5

Intraspecies BC method-generated Mesp1/2-DKO mouse model (A) Schematic of intraspecies blastocyst complementation (BC) method. Red fluorescent protein (RFP) expressing mouse WT ESCs were injected into the embryos obtained by crossing Mesp1/2 double heterozygous (DHet) male and female mice. WT ESCs+Mesp1/2-DKO or WT ESCs+non-Mesp1/2-DKO chimeras were dissected at E14.5. (B) Genotype of Mesp1/2-DKO, Mesp1/2-DHet, and WT embryos. RFP negative population was sorted to remove WT cells derived from injected ESCs (RFP+). See genotyping strategy in Fig.S3E. (C) Representative image of embryos and organs derived from WT ESCs (RFP+) +Mesp1/2-DKO or WT ESCs (RFP+) +non-Mesp1/2-DKO chimeras. Chimeras were viable in the Mesp1/2-DKO genotype, and the RFP-expressing heart was observed. (T; Tail, K: Kidney, G: Gonad, B: Bladder, H: Heart, Li: Liver, Lu: Lung, S: Stomach, I: Intestine) Scale bars, 1 mm. (D) Flow cytometry analysis of organs (forelimb, heart, intestine, lung, stomach, kidney, and tail) in chimeras from WT ESCs (RFP+) +Mesp1/2-DKO or WT ESCs (RFP+) +non-Mesp1/2-DKO. The heart is almost composed of RFP-expressing WT cells in all embryos of the Mesp1/2-DKO genotype (n = 3). (E) Results of intraspecies blastocyst complementation by injecting mouse ESCs into blastocysts obtained by crossing Mesp1/2-DHet with Mesp1/2-DHet. Chimeras with almost 100% ESC contribution were removed from the genotype analysis owing to the difficulty of sorting RFP negative population.

Figure 6

Interspecies BC method for generating Mesp1/2-DKO mouse model (A) Schematic of interspecies blastocyst complementation (BC) method. Green fluorescent protein (GFP) expressing rat ESCs were injected into the embryos obtained by crossing Mesp1/2 double heterozygous (DHet) male and female mice. Rat ESCs+Mesp1/2-DKO or rat ESCs+non-Mesp1/2-DKO chimeras were dissected at E14.5 and E12.5. (B) Results of interspecies blastocyst complementation by injecting rat ESCs into blastocysts obtained by crossing Mesp1/2-DHet with Mesp1/2-DHet. The numbers in parentheses indicate resorbed embryos. (C) Representative images of embryos and hearts derived from chimeras from rat ESCs+Mesp1/2-DKO or rat ESCs+non-Mesp1/2-DKO. The chimeras were viable in the Mesp1/2-DKO genotype, and GFP expressing heart was observed. Scale bars, 1 mm. (D) Quantitative real-time PCR results for mouse Myl2, Myl7, Aldh1a2, Pecam1, and Postn. Data were normalized to mouse Gapdh expression levels. Samples were extracted from the hearts of chimeras. All values are expressed as mean ± standard deviation from at least triplicate experiments (DKO: n = 3 rat chimeras, non-DKO: n = 7 rat chimeras). ∗∗∗: p < 0.01; unpaired two-tailed Student’s t test. (E) Variation in the presence of rat cells in each cardiac tissue component. Mostly, rat Myl2, Myl7, Aldh1a2, Pecam1, and Postn were detected compared with mouse genes in the rat Mesp1/2-DKO chimera genotype. Open circles in the non-Mesp1/2-DKO genotype indicate the results obtained from higher rat chimerism in the tail (28–67%). Open triangles in the non-Mesp1/2-DKO genotype showed the results obtained from lower rat chimerism in the tail (6–12%). All values are expressed as mean ± standard deviation from at least triplicate experiments (n = 3 in DKO; n = 7 in non-DKO with higher rat chimerism; n = 6 in non-DKO with lower rat chimerism).

Figure 7

Analysis of mouse-rat chimera in non-Mesp1/2-DKO mouse (A) Flow cytometry analysis of tail of rat ESCs+non-Mesp1/2-DKO chimeras at E12.5 and E14.5. (n = 17 at E12.5, n = 13 at E14.5). (B) Flow cytometry analysis of tail, heart, and lung of rat ESCs+non-Mesp1/2-DKO chimeras at E12.5 (n = 8). (C) Representative heart images of higher rat chimerism (44.2%, 44.2%, and 54.7% in tail) and lower rat chimerism (11.7%, 8.3% in tail) at the E12.5 stage. No rat contribution was observed at E12.5 and E10.5 stages in the non-Mesp1/2-DKO genotype. The arrow indicates the interventricular sulcus. The white arrow indicates the loss of rat cells' contribution to the left ventricle. (Scale bar: 1 mm). (D) Representative embryo, heart, and HE staining images of rat chimera in the non-Mesp1,2-DKO genotype (Scale bar: 1 mm).