Generation of CD34 + Cells from CCR5-Disrupted Human Embryonic and Induced Pluripotent Stem Cells

Abstract

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However, Δ32 donors are scarce, heterologous bone marrow transplantation is not exempt of risks, and genetic engineering of autologous hHSCs is not trivial. Here, we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34⁺ cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS.

Introduction

T o date, there is no universal cure for AIDS. Antiretroviral therapies can control viral replication, but this does not eradicate the infection, and viral rebound normally occurs when drugs are withdrawn (Richman et al., 2009). C-C chemokine receptor type 5 (CCR5) is a co-receptor for HIV-1 on the surface of target cells (Deng et al., 1996). Individuals with the natural homozygous CCR5 Δ32 allele, which bears a 32-bp deletion within the coding region that results in a frame shift, are resistant to HIV-1 infection (Liu et al., 1996). Small-molecule CCR5 antagonists can block HIV-1 entry into target cells and have been used successfully in clinical trials (Fätkenheuer et al., 2005), but this approach can produce resistance by selection pressure (Kuhmann et al., 2004). Allogenic transplantation of Δ32 human hematopoietic stem cells (hHSCs) were able to control HIV-1 infection for up to 20 months without viral rebound after withdrawing antiretroviral therapy (Hütter et al., 2009) and have been shown to cure the infection as well (Allers et al., 2010). However, application of this procedure is limited due to rare availability of Δ32 donors (Martinson et al., 1997). Autologous transplantation using genetically modified hHSCs potentially offers an alternative method (Cui et al., 2003). Zinc finger nucleases (ZFNs) that specifically disrupt the CCR5 gene have already been used in CD4⁺ T cells and CD34⁺ hHSCs, conferring resistance to HIV-1 in vitro and in vivo (Perez et al., 2008; Holt et al., 2010). Importantly, HIV-1 infection prior to ZFN engineering of autologous T cells or hHSCs from AIDS patients cannot be excluded and could create a viral reservoir (Carter et al., 2010). An interesting surrogate is to derive hHSCs from genetically modified hiPSCs (Hockemeyer et al., 2009; Zou et al., 2009) produced from healthy tissues of HIV-1 infected patients. CCR5 disruption of human embryonic stem cells (hESCs) by ZFNs has been reported recently using a baculovirus-based system, though hematopoietic differentiation was not shown (Lei et al., 2011). To the best of our knowledge, our work is the first to report CCR5-disrupted hHSCs from hiPSCs. Our results suggest that the future generation of CCR5-disrupted hHSCs from ZFN-modified patient specific hiPSCs might be a feasible option for treating AIDS.

Materials and Methods

Cell culture and characterization

H9 hESCs (passage 47–56, WiCell Research Institute) and an hiPSC clone produced from skin fibroblasts from a healthy human male and fully characterized (passage 15–26, Esteban et al., 2010) were maintained in a feeder-free culture system using mTesR1 (Stem Cell Technologies) and Matrigel (BD Bioscience). OP9 cells (ATCC) were cultured on gelatinized six-well dishes (2×10⁴ per well) in OP9 growth medium consisting of α-modified minimum essential media (Invitrogen) supplemented with 20% non–heat-inactivated defined fetal bovine serum (HyClone). Alkaline phosphatase (AP) staining, reverse-transcription polymerase chain reaction (RT-PCR) and karyotyping were performed following standard procedure. For teratomas, 2×10⁶ cells were injected into NOD-SCID mice.

CCR5 gene modification

ZFNs disrupting the human CCR5 locus were previously described (Perez et al., 2008). The corresponding ZFN sequences were cloned in one open reading frame and linked by a 2A sequence (Szymczak et al., 2004); the expression was controlled by a CAG promoter (Alexopoulou et al., 2008). The plasmid for homologous recombination was prepared by using the pUC19 vector. Enhanced green fluorescence protein (EGFP) and a puromycin-resistance cassette were linked by a 2A sequence as well, with the expression controlled by the EF1α promoter, and cloned within two homologous recombination arms. FuGENE HD transfection reagent (Roche Applied Science) was used for delivery. Puromycin (Sigma-Aldrich) was added at a concentration of 1 μg/mL.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde and washed. The coverslips were blocked in 4% goat serum in phosphate-buffered saline before processing. Nuclei were stained with 4′,6-diamidino-2-phenylindole (Sigma). A Leica DMI4000B microscope (Leica Microsystems) was used for detection. All primary and secondary antibodies of this study are listed in Supplementary Table S1 (Supplementary Data are available online at www.liebertonline.com/hum).

Hematopoietic differentiation

We adopted a protocol from Choi et al. (2009). Briefly, OP9 cells were grown for 8 days with half-medium changes on day 4. Undifferentiated hESCs and hiPSCs were added (∼2×10⁵) after treatment with 1 mg/mL dispase (Invitrogen) and dispersion of the cultures into small clumps by scraping and pipetting. The co-cultures were incubated for up to 12 days at 37°C in normoxic conditions and 5% CO₂, with half-medium changes every other day from day 4. To obtain a single-cell suspension, the cultures were treated with collagenase IV (Sigma), followed by trypsin/EDTA (0.05%). The suspension was further disrupted by pipetting and filtered through a 70-μm strainer before use in fluorescent activated cell sorter (FACS) analysis with FACSArial flow cytometer (BD Bioscience) or clonogenic forming cell (CFC) assays. For CFC assays, CD34⁺ cells were isolated using magnetic-activated cell sorting with a human CD34 Microbeads kit (Miltenyi Biotec) and added (2×10³/mL) to low-adherence six-well plates (Stem Cell) using MethoCult GF+ H4435 semisolid medium (Stem Cell).

Results and Discussion

To disrupt the human CCR5 locus in hiPSCs, we employed an expression vector in which the ZFN sequences were linked by a 2A sequence and a donor vector for homologous recombination that produced puromycin resistance and EGFP (Fig. 1A). We also used hESCs to compare whether the maintenance of pluripotent characteristics after the gene editing approach is similar in both cell types. In this regard, one could argue that the artificial pluripotent state of hiPSCs might become unstable after zinc finger manipulation. Two days post-transfection EGFP was visualized and the medium was changed to selection medium containing puromycin. Resistant colonies emerged after another 4 days and puromycin was maintained for at least six passages. We then performed PCR analysis using primers that amplified the recombination cassette (Fig. 1A), and we found integration in 67% of the hiPSC and hESC colonies (Fig. 1B). We chose a positive hiPSC colony and another one for hESCs, hereafter termed ΔR5 cells, for further study (Fig. 1C). PCR using additional primers (Fig. 1A) confirmed site-specific integration but also demonstrated that the ΔR5 hiPSC and ΔR5 hESC colonies contained cells homozygous or heterozygous for the CCR5 deletion (Fig. 1D). The two colonies (passage 14 for hiPSCs and 19 for hESCs at the moment of analysis) retained a normal karyotype (Supplementary Fig. S1), expressed hESC-specific genes to the level of normal hESC and hiPSC colonies (Fig. 1E), stained positive for AP and other hESC-surface markers (Fig. 1F), and formed teratomas (Fig. 1G). Next, we investigated whether these modified pluripotent cell lines had the potential to be differentiated into CD34⁺ cells. FACS analysis confirmed the appearance of CD34⁺ cells with dynamics similar to those of normal hESCs and hiPSCs (Fig. 2A). We also noticed that EGFP expression was divided into high- and low-expressing populations, both in the CD34⁺ cells and the remaining cells (Fig. 2B). This is in agreement with the observation that the ΔR5 hiPSC colony is a combination of homozygous and heterozygous CCR5-deleted cells; PCR analysis for purified CD34⁺ cells confirmed this finding (Fig. 2C). Next, we characterized the multi-differentiation potential of ΔR5 CD34⁺ cells derived from hiPSCs. Addition of a cocktail of cytokines to purified CD34⁺ cells cultured on semisolid medium produced different types of colonies: CFC macrophage, CFC granulocyte, CFC granulocyte/macrophage, CFC granulocyte/erythrocyte/macrophage/megakaryo-cyte, and CFC erythroid (Fig. 2D).

FIG. 1.

Disruption of the endogenous C-C chemokine receptor type 5 (CCR5) locus in human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) by zinc-finger nuclease (ZFN)-mediated homologous recombination. (A) Scheme of CCR5 gene disruption using ZFNs that target the coding sequence within exon 3. A pair of CCR5-specific ZFNs was linked using 2A sequences and cloned into an expression vector controlled by the CAG promoter. L and R indicate left and right relative to the cutting point, respectively; Puro^R means puromycin resistance. The donor vector (for homologous recombination) contained an EF1α-EGFP-Puro-polyA expression cassette flanked by two arms of CCR5 homologous sequences (2.2 kb each). The position of the polymerase chain reaction (PCR) primers (Supplementary Table S2) used to confirm the targeted insertion is also indicated. (B) PCR screening of CCR5-disrupted colonies with primers 1 and 2. (C) Immunofluorescence microscopy of a putative ΔR5 colony stably expressing enhanced green fluorescence protein (EGFP). Scale bars are 200 μm. (D) More-detailed colony PCR analysis using primers indicated in A. Amplification of both endogenous and putative homologous recombination (HR) production can be observed in ΔR5 colonies; only endogenous production was observed in normal colonies. (E) Reverse-transcription PCR (RT-PCR) shows expression of hESC-specific mRNAs in ΔR5 hESCs and ΔR5 hiPSCs to the level of wild-type hESCs and hiPSCs, in contrast with human foreskin fibroblasts (HFFs). (F) AP staining and immunofluorescence for the indicated hESCs markers (in red). Nuclei are stained in blue with 4′,6-diamidino-2-phenylindole; enhanced green fluorescence protein (EGFP) is expressed from the HR cassette. Scale bars are 200 μm. (G) Hematoxylin and eosin staining of sections of a teratoma formed using ΔR5 H9 hESCs shows structures corresponding to ectodermal, mesodermal, and endodermal lineages. Tumors were excised 8–10 weeks later, fixed, embedded in paraffin, and sectioned. Scale bars are 400 μm. Color images available online at www.liebertonline.com/hum

FIG. 2.

Directed differentiation of CCR5-disrupted hiPSCs and hESCs into CD34⁺ cells. (A) Flow cytometry analysis of ΔR5 and normal hiPSCs and hESCs co-cultured with OP9 cells shows progressive increase in CD34⁺ cells from day 2 to 12. (B) Scatter plots of a similar experiment using ΔR5 hiPSCs. (C) The two groups of CD34⁺ cells (high or low EGFP) from ΔR5 hiPSCs were analyzed by PCR using selected primers as in Fig. 1A. CD34⁺ cells from normal hiPSCs are also included. (D) CD34⁺ cells derived from ΔR5 hiPSCs produced all different types of CFCs. Photographs were taken after 14–21 days. Scale bars are 100 μm. CFC-M, CFC macrophage; CFC-G, CFC granulocyte; CFC-GM, CFC granulocyte/macrophage; CFC-GEMM, granulocyte/erythrocyte/macrophage/megakaryo-cyte; CFC-E, CFC erythroid. Color images available online at www.liebertonline.com/hum

In summary, here we have shown the generation of CD34⁺ cells from CCR5-deleted hiPSCs and hESCs. These cells had the potential to be differentiated into different types of blood cell types. In the future, a similar approach could provide a stable supply of autologous CCR5-disrupted CD34⁺ cells that might be useful for transplantation in AIDS patients. Before this possibility can be considered, several relevant concerns would need to be addressed. Notably, hiPSCs need to be generated without exogenous factor integration (Yu et al., 2009; Warren et al., 2010) and tested for exclusion of somatic point mutations or epigenetic modifications (Bock et al., 2011; Gore et al., 2011). Generation of hiPSCs in a feeder-free and chemically defined medium (Yu et al., 2011) is also important for guaranteeing the exclusion of xeno-contaminants. Likewise, a feeder-free hematopoietic differentiation system, based on embryoid body formation (Galić et al., 2009), would be necessary to eliminate the need for murine cocultures. On the other hand, the combination of homozygous and heterozygous CCR5-deleted cells within the same colony may not be a critical issue because once these cells have reached macrophage and lymphoid lineages, only those cells bearing homozygous deletion would survive HIV-1 selective pressure in vivo (Holt et al., 2010). Besides, single-cell cloning using the Rho-associated kinase inhibitor could solve this problem (Watanabe et al., 2007). Despite the current limitations, the model presented here may be sufficient for creating humanized mice models for the study of HIV-1 infection (Galić et al., 2006; Holt et al., 2010; Zhang et al., 2007). Such study could be a first step towards considering clinical application of induced pluripotent stem cells in AIDS therapy.

Footnotes

Acknowledgements

The authors would like to thank Dajiang Qin, Dr. Duanqing Pei, Yongxiang Yan, Cuizhu Huang, Hui Zhang, Dr. Huaqiang Yang, and the staff in the Public Instrument Center and Animal Center for discussions and experimental/technical support. This work was supported by a research grant from the Chinese Academy of Sciences (KSCX1-YW-10).

Author Disclosure Statement

No competing financial interests exist.

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