Highlights
- •4CL medium and MYCN/BCL2 improve interspecies integration capacity of human iPSCs
- •SIX1−/− SALL1−/− pig embryos provide a nephric-defective niche for iPSC-derived cells
- •Human 4CL/N/B iPSCs produce a humanized mesonephros in nephric-defective pig embryos
- •Human 4CL/N/B iPSCs have limited contribution to other organs in pig embryos
Summary
Heterologous organ transplantation is an effective way of replacing organ function but is limited by severe organ shortage. Although generating human organs in other large mammals through embryo complementation would be a groundbreaking solution, it faces many challenges, especially the poor integration of human cells into the recipient tissues. To produce human cells with superior intra-niche competitiveness, we combined optimized pluripotent stem cell culture conditions with the inducible overexpression of two pro-survival genes (MYCN and BCL2). The resulting cells had substantially enhanced viability in the xeno-environment of interspecies chimeric blastocyst and successfully formed organized human-pig chimeric middle-stage kidney (mesonephros) structures up to embryonic day 28 inside nephric-defective pig embryos lacking SIX1 and SALL1. Our findings demonstrate proof of principle of the possibility of generating a humanized primordial organ in organogenesis-disabled pigs, opening an exciting avenue for regenerative medicine and an artificial window for studying human kidney development.
Introduction
The shortage of immunohistocompatible donors for organ transplantation is a universal problem with no direct solution. A promising alternative is to generate viable interspecies chimeras involving large mammals bearing human tissues through embryo complementation with pluripotent stem cells (PSCs), which could take over a particular organogenesis-disabled niche in the host made by gene editing. Interspecies organogenesis has been successfully achieved between mice and rats for organs such as the pancreas, thymus, and the kidney. Pigs are a highly attractive target for growing human organs given their similarities with humans in physiology and organ size, as well as in embryonic development. However, obtaining a high degree of chimerism with mammalian species beyond rodents has been challenging. Although, human endothelium and patches of skeletal muscle tissue have been generated inside ETV2 or MYF5/MYOD/MYF6-null pig embryos, no solid organs have been produced to date because of the overall poor contribution of human PSC-derived cells to porcine tissues.
There are two major reasons for the low interspecies chimerism: (1) the competition with donor cells in the blastocyst and later in the tissue niche; and (2) differences in the developmental state between the donor human PSC-derived cells and the host cells, which prevents synchronized developmental progression.
Activation of pro-survival pathways in the injected PSCs can be used to boost chimerism by antagonizing the apoptosis induced by cell competition. In this regard, we report that the combination of MYCN and BCL2 (N/B) greatly enhances intra-niche competitiveness in mouse, rabbit, and pig embryos, compared with single-factor overexpression. Moreover, we recently developed a new culture medium allowing for the stepwise rolling back of human PSCs to earlier developmental stages, without noticeable problems of genome stability in the conversion process. This medium (4CL) allowed up to 1% human-mouse chimerism at embryonic day 10.5 (E10.5), which is comparable to the integration efficiency of primed PSCs overexpressing anti-apoptotic factors.
The human kidney is one of the most transplanted solid organs worldwide and also one of the earliest appearing during embryogenesis. It undergoes sequential pronephros, mesonephros, and metanephros specification. The formation and regression of the pronephros and then mesonephros are well orchestrated and are followed by invasion of the metanephric mesenchyme by the ureteric bud, which initiates metanephros formation. Studies of renal development in different mammals have demonstrated that SIX1 regulates mesonephric tubule (mesonephroi) formation and ureteric bud branching for metanephrogenesis, while SALL1 maintains nephron progenitors and nascent nephrons in the metanephric mesenchyme. Hence, we posited that engineering SIX1/SALL1-null pig embryos would produce a nephric-defective niche from early mesonephros to late metanephros, and this could allow human PSCs to fill in the whole renal development program. We also envisaged that a combination of N/B overexpression and optimized PSC culture conditions could enable a superior degree of chimerism after embryo complementation and thus help produce a human kidney primordium in these organogenesis-disabled pigs. To further optimize high xeno-chimera efficiency, we also addressed other potential bottlenecks such as the blastocyst complementation procedure.
Results
Generation of human 4CL/N/B PSCs with superior chimeric potential
Recent studies have shown that the developmental state of human PSCs is relevant for successful chimera formation, and human PSCs exhibit a spectrum of pluripotency varying with regard to the culture media. These differences led us to study human PSCs cultured in the well-characterized primed culture medium mTeSR1 and several naive PSC media. The latter included our recently reported 4CL, RSeT (a commercial medium based on NHSM), PXGL, and 5iLA. We compared the gene expression pattern of previously reported single-cell RNA sequencing (scRNA-seq) datasets of PSCs cultured in these conditions with human embryo scRNA-seq using uniform manifold approximation and projection (UMAP) representation and Pearson correlation analysis. This showed that 4CL PSCs were more homogeneously clustered close to E5 of the human pre-implantation blastocyst than other naive PSCs and primed PSCs (Figures S1A–S1C). This result was confirmed by examining the expression of specific naive and primed pluripotency genes (Figure S1D). We also integrated 4CL PSCs with both pre-implantation and post-implantation human embryo datasets, which consistently showed a good match with the E5 blastocyst (Figures S1E and S1F).
Next, we screened for conditions allowing normal blastocyst formation after injecting DsRed-labeled induced PSCs (iPSCs) in 4CL medium (4CL iPSCs) into early parthenogenetic pig embryos. The optimal protocol comprised the administration of three to five iPSCs into morula or early blastocyst-stage pig embryos followed by culture in a medium composed of porcine zygote medium (PZM) and 4CL medium in a 1:1 ratio (Figures S2A–S2F). Using these conditions, we systematically compared the chimeric competency of 4CL iPSCs in blastocyst with that of the same iPSC clone grown in other naive PSC media and mTeSR1 (Figure 1A). By quantifying the ratio of DsRed+ area relative to the blastocyst area, we found that 4CL iPSCs attained the highest chimera formation capacity at the blastocyst stage (Figures 1B, 1C, and S2G–S2I). To further enhance the chimeric capacity, we overexpressed N/B in 4CL iPSCs using a doxycycline-inducible system (Figures S2J and S2K), observing that this combination (4CL/N/B) yielded the highest degree of chimeric competency assessed by quantification of the DsRed+ area (Figures 1D and 1E). Quantification of the average number of DsRed+ human cells in each blastocyst confirmed that 4CL/N/B iPSCs exhibit the highest chimera competency (Figure 1F), consistent with the DsRed+ area analysis. Of note, 4CL/N/B PSCs had a transcriptional profile enriched in naive pluripotency genes similar to 4CL PSCs (Figures 1G, 1H, and S2L). However, we observed an enrichment of cell-cycle-related gene ontology (GO) terms for upregulated genes in 4CL/N/B PSCs and a higher percentage of cells entering the G2/M phase compared with 4CL alone (Figures 1I and 1J). This indicated that N/B enhances PSC proliferation but does not affect the pluripotency state endorsed by 4CL medium.
