Assessment of safety and immunogenicity of MHC homozygous iPSC-derived CD34+ hematopoietic progenitors in an NHP model

Abstract

Administration of ex vivo expanded somatic myeloid progenitors has been explored as a way to facilitate a more rapid myeloid recovery and improve overall survival after myeloablation. Recent advances in induced pluripotent stem cell (iPSC) technologies have created alternative platforms for supplying off-the-shelf immunologically compatible myeloid progenitors, including cellular products derived from major histocompatibility complex (MHC) homozygous superdonors, potentially increasing the availability of MHC-matching cells and maximizing the utility of stem cell banking. However, the teratogenic and tumorigenic potential of iPSC-derived progenitor cells and whether they will induce alloreactive antibodies upon transfer remain unclear. We evaluated the safety and efficacy of using CD34+CD45+ hematopoietic progenitors derived from MHC homozygous iPSCs (iHPs) to treat cytopenia after myeloablative hematopoietic stem cell (HSC) transplantation in a Mauritian cynomolgus macaque (MCM) nonhuman primate (NHP) model. We demonstrated that infusion of iHPs was well tolerated and safe, observing no teratomas or tumors in the MCMs up to 1 year after HSC transplantation and iHP infusion. Importantly, the iHPs also did not induce significant levels of alloantibodies in MHC-matched or -mismatched immunocompetent MCMs, even after increasing MHC expression on iHPs with interferon-γ. These results support the feasibility of iHP use in the setting of myeloablation and suggest that iHP products pose a low risk of inducing alloreactive antibodies.

Graphical abstract

Figure 1.

Characterization of iHPs. (A) Schematic diagram of HSPC transplantation and iHP infusion experiments. (B) Timeline for experiments. (C) Representative flow cytometric dot plot and histograms showing expression of CD34 and CD90 (filled histogram) vs unstained control (empty histogram in iHPs; cells are gated on live CD45⁺ cells). (D) Colony-forming cell (CFC) potential of iHPs. Results are mean ± standard deviation for 3 independent experiments. (E) Representative flow cytometric experiment showing MHC class I and class II expression (filled histograms) relative to unstained controls (empty histogram) in iHPs before and after activation with interferon-γ (IFN-γ). Cells are gated on live CD45⁺ cells. LV, lentivirus; PB, peripheral blood.

Figure 2.

Complete blood counts to monitor hematopoietic reconstitution. Results are mean ± standard deviation of 3 different animals in each group. abs, absolute counts; ths/μL, thousands/μL.

Figure 3.

Detection of iPSC-derived cells in peripheral blood. (A) Percentage of eGFP⁺ or tdTomato⁺ cells within granulocyte or monocyte forward scatter (FSC) vs side scatter (SSC) gate after gating on live single cells in individual animals. (B) Representative dot plots showing eGFP or tdTomato vs CD11b or CD14 expression by cells within granulocyte or monocyte FSC vs SSC gate. (C) Genomic PCR analysis using primers specific for eGFP or tdTomato to detect iPSC-derived cells in the peripheral blood. Peripheral blood mononuclear cells from animal undergoing transplantation with eGFP-transduced HSPCs were used as positive control for animals that received eGFP-tagged iHPs. iHPs expressing tdTomato were used as positive control for animals that received iHPs tagged with tdTomato. Peripheral blood collected before transplantation was used as negative control.

Figure 4.

Detection of eGFP- or tdTomato-tagged iPSC-derived cells by genomic PCR in biopsy specimens collected from BM, colon, and lymph nodes. BM, lymph node, and colon biopsies collected from animal that did not receive any eGFP or tdTomato cells were used as negative control. Peripheral blood mononuclear cells from animal undergoing transplantation with eGFP-transduced HSPCs were used as positive control for animals that received eGFP-tagged iHPs. Day-10 iHPs expressing tdTomato were used as positive control for animals that received iHPs tagged with tdTomato.

Figure 5.

Detection of eGFP- or tdTomato-tagged iPSC-derived cells by genomic PCR in collected postmortem tissues. Peripheral blood mononuclear cells from animal undergoing transplantation with eGFP-transduced HSPCs were used as positive control for animals that received eGFP-tagged iHPs. Tissues were collected 25 days (cy0959) or 1 year (remaining animals) after iHP infusion. Day-10 iHPs expressing tdTomato were used as positive control for animals that received iHPs tagged with tdTomato.

Figure 6.

Evaluation of immunogenicity of iHPs derived from MHC homozygous iPSCs. (A) Schematic diagram for the assessment of alloimmune response to iPSC-derived iHPs. (B) Titration of serum from MCMs infused with MHC-mismatched peripheral blood lymphocytes (positive control). Day-0 serum before lymphocyte infusion. Day-90 serial dilutions of serum collected 90 days after infusion. nMFI values and fluorescence-activated cell sorting plots are shown. (C) Titration graphs of serum samples from each group collected at different time points from animals that were infused with MHC homozygous iHPs that were not activated with IFN-γ. (D) Titration graphs of serum samples from each group collected at different time points from animals that were infused with MHC homozygous iHPs that were activated with IFN-γ and analyzed. Dot plots of 1/20 and 1/40 dilutions of serum collected at different times after iHP infusion from representative animals are shown. In panels C and D titration graphs, each dot represents a different animal from each group. The different days of the serum collection after the first iHP infusion are indicated by different colors (day 0-120).