Human post-implantation blastocyst-like characteristics of Muse cells isolated from human umbilical cord

Abstract

Muse cells, identified as cells positive for the pluripotent surface marker SSEA-3, are pluripotent-like endogenous stem cells located in the bone marrow (BM), peripheral blood, and organ connective tissues. The detailed characteristics of SSEA-3(+) cells in extraembryonic tissue, however, are unknown. Here, we demonstrated that similar to human-adult tissue-Muse cells collected from the BM, adipose tissue, and dermis as SSEA-3(+), human-umbilical cord (UC)-SSEA-3(+) cells express pluripotency markers, differentiate into triploblastic-lineage cells at a single cell level, migrate to damaged tissue, and exhibit low telomerase activity and non-tumorigenicity. Notably, ~ 20% of human-UC-SSEA-3(+) cells were negative for X-inactive specific transcript (XIST), a naïve pluripotent stem cell characteristic, whereas all human adult tissue-Muse cells are XIST-positive. Single-cell RNA sequencing revealed that the gene expression profile of human-UC-SSEA-3(+) cells was more similar to that of human post-implantation blastocysts than human-adult tissue-Muse cells. The DNA methylation level showed the same trend, and notably, the methylation levels in genes particularly related to differentiation were lower in human-UC-SSEA-3(+) cells than in human-adult tissue-Muse cells. Furthermore, human-UC-SSEA-3(+) cells newly express markers specific to extraembryonic-, germline-, and hematopoietic-lineages after differentiation induction in vitro whereas human-adult tissue-Muse cells respond only partially to the induction. Among various stem/progenitor cells in living bodies, those that exhibit properties similar to post-implantation blastocysts in a naïve state have not yet been found in humans. Easily accessible human-UC-SSEA-3(+) cells may be a valuable tool for studying early-stage human development and human reproductive medicine.

Keywords: DNA methylation; Human post-implantation blastocyst; Muse cells; Single-cell RNA sequencing; Umbilical cord; XIST.

Fig. 1

Characterization of SSEA-3(+) cells from h-UC-MSCs. A Expression of CD133 and HLA-G in h-UC-MSCs, h-BM-MSCs, h-ADSCs, and NHDF. B Clusters formed in single-cell suspension culture from h-UC-SSEA-3(+) cells. These clusters expressed Nanog, Oct3/4, Sox2, PAR4, Tra-1-81. C Telomerase activity in HeLa cells, h-UC-SSEA-3(+) cells, and non-template control (NTC) measured by ddPCR. Each dot on the ddPCR output represents a unique droplet that is either positive or negative for a fluorescent signal whose threshold was 6000. D SCID mouse testes injected with MEF (3 months), mouse ESCs (3 months), PBS (6 months), and h-UC-SSEA-3(+) cells (6 months). E In vitro migration of h-UC-SSEA-3(+) cells and h-BM-Muse cells toward damaged liver tissue slice. F In vivo dynamics of intravenously injected h-UC-SSEA-3(+) cells and h-BM-Muse cells in the acute liver injury model. One week after intravenous administration, the Akaluc signal was detected predominantly in the liver while it was stronger in the h-UC-Muse group than in the h-BM-Muse group. G Total flux by ex vivo imaging. In organs other than the liver and lung, the Akaluc signal was under the detection limit. H Expression of XIST in h-UC-, h-BM-, h-AT, and h-DT-Muse cells

Fig. 2

Comparison of human Muse cells and h-embryo cells in scRNA-seq. A UMAP of h-UC-, h-BM-, h-AT, h-DT-Muse cells, integrated with published datasets of h-embryo (E3-7) [38], h-embryo (E8-14) [38], and h-embryo (E16) cells [39]. B The enlarged image of UMAP of h-UC-Muse cells and h-embryo cells in the square of (A). C The ratio of h-UC-Muse cells, h-embryo (E3-7), h-embryo (E8-14), and h-embryo (E16) cells in clusters 1–16 classified in (B). D Dot plots of the correlation coefficient for cell types of h-embryos in clusters 1, 3, and 7 of h-UC-Muse cells. The -log10 (p-value) is indicated by the color intensity. The dot size indicates the correlation coefficient. E GO term of commonly upregulated genes in each cell type (clusters 1, 3, 7 in h-UC-Muse cells, as well as epiblast, trophectoderm, primitive endoderm, primitive streak, and advanced mesoderm with a high correlation coefficient among them) compared with the remaining h-embryo cells. F GO term of commonly downregulated genes in each cell type (clusters 1, 3, 7 in h-UC-Muse cells, as well as epiblast, trophectoderm, primitive endoderm, primitive streak, and advanced mesoderm with a high correlation coefficient among them) compared to the remaining h-embryo cells. G Heatmap of the lineage-specific marker genes in each type of h-embryo and h-Muse cell. H Violin plot showing the expression levels of DSG2, DSC3, and plakoglobin in h-UC-, h-BM-, h-AT, and h-DT-Muse cells

Fig. 3

Comparison of h-Muse cells and h-embryo cells in enzymatic methyl sequencing. A Methylation landscape of h-Muse cells and h-embryo cells. The averaged DNA methylation levels were calculated based on the overlapped 100-bp tiles detected in h-Muse cells and h-embryo cells. B Heatmap with hierarchical clustering used a distance metric based on Pearson’s correlation coefficients and average linkage in h-Muse cells and h-embryo cells. C GO term of genes with low methylation levels (< 10%) in both h-UC-Muse cells and h-post-implantation blastocysts. D GO term of hypomethylated genes with relative methylation levels lower than 30% in h-UC-Muse cells compared with h-adult tissue-Muse cells. E GO term of hypermethylated genes with relative methylation levels higher than 30% in h-UC-Muse cells compared with h-adult tissue-Muse cells. F Heatmap of the lineage-specific marker genes in h-Muse cells. Values of h-UC-Muse cells were set as 1

Fig. 4

In vitro differentiation of h-UC-Muse cells into extraembryonic-lineage marker(+) cells. A Expression of extraembryonic cell-lineage genes in the 4 types of naïve Muse cells, as well as in those from day 3 to 3 weeks after induction (normalized by ACTB). JEG3 was set as a positive control (P.C.). Values of JEG3 were set as 1. B Extraembryonic-lineage induction of h-Muse cells. Human-UC-Muse cells at 3 weeks after induction contained multinucleated cells in phase contrast and β2-microglobulin (used to visualize cell morphology) immunostaining. C Immunocytochemistry for extraembryonic cell markers (HLA-G, hCGA, GABRP, and ERVW-1) in the 4 types of h-Muse cells at 3 weeks after induction. D Western blot of hCGB, a functional marker of human trophoblasts, in the naïve state and 3 weeks after induction in h-UC-Muse cells. JEG3 was set as a positive control (P.C.). Bars: 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. ND not detected

Fig. 5

In vitro differentiation of h-UC-Muse cells into germline-lineage marker(+) cells. A Expression of genes related to germline lineage in the 4 types of naïve Muse cells, as well as in those from day 4 to day 6 after induction (normalized by ACTB). PGCLC was set as a positive control (P.C.). Values of PGCLC were set as 1. B Immunocytochemistry for germline markers (BLIMP1, SOX17, and NANOS3) in the 4 types of h-Muse cells at 3 weeks after induction. Bars: 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. ND not detected

Fig. 6

In vitro differentiation of h-UC-Muse cells into hematopoietic-lineage marker(+) cells. A Expression of genes related to the hematopoietic lineage in the 4 types of naïve Muse cells, as well as those from day 3 to 3 weeks after induction (normalized by ACTB). For the positive control, human fetus whole total RNA (TIE2, FLK-1, CD31, and PTPRC) and human blood total RNA (c-KIT and CD34) were used (P.C.). Values of PGCLC were set as 1. B Immunocytochemistry for FLK-1, c-Kit, and CD34 in the 4 types of h-Muse cells at 3 weeks after induction. C Western blot of CD201 and ITGA3 in the naïve state and 3 weeks after induction in h-UC-Muse cells. HeLa cells were set as a positive control (P.C.), according to previous reports [116, 117]. Bars: 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001. ND not detected