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Research Highlights

Sharma, Monal PhD1; Luo, Xunrong MD, PhD1

doi: 10.1097/TP.0000000000002884
In View: Research Highlights
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1 Division of Nephrology, Department of Medicine, Duke University School of Medicine, Durham, NC.

Received 12 July 2019.

Accepted 12 July 2019.

The authors declare no funding or conflicts of interest.

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Generation of Hypoimmunogenic Human Pluripotent Stem Cells

Han X, Wang M, Duan S, et al. Proc Natl Acad Sci U S A, May 2019

A pragmatic obstacle in individualized cell therapy is the enormous regulatory burden for the generation and quality control of patient-specific cellular products. The use of “off-the-shelf” banked cells from one or defined sources of individuals is therefore highly attractive.1 This approach, however, presents a different challenge of alloimmune responses resulting from immunological disparities between the donors of banked cells and the ultimate recipients of the cellular products. In addressing this challenge, Han et al2 generated hypoimmuneogenic human pluripotent stem cells (hPSCs) by employing a multiplex CRISPR/Cas9 genome editing technology to minimize both adaptive and innate immune responses.

Using a dual guide multiplex strategy in the hPSC cell line HUES8, the authors excised the human leukocyte antigen (HLA)-A/-B/-C genes from the genome of hPSCs. Next, they targeted the CIITA transcription regulator to eliminate HLA class II gene expression. Deletions of these polymorphic HLA class I and II molecules would in theory eliminate T cell-mediated adaptive immune responses towards the hPSC products. To control innate immune responses, the authors engineered HLA-G minimizing NK-mediated lysis, CD47 to minimize macrophage-mediated phagocytosis, and PD-L1 to facilitate PD-1–mediated immune suppression. The 3 genes were knocked in to the AAVS1 safe harbor locus, thus minimizing random integration and off-target effects. In an in vitro assay of T cell–mediated immune responses, modified hPSCs exhibited ~50% reduction of T-cell proliferation, activation, and CD8+ T-cell–mediated cytotoxicity. The diminished adaptive immune response was also recapitulated in an in vivo assay measuring hPSC-derived teratoma formation in immunodeficient mice reconstituted with human CD8+ T cells. Lastly, knock-in of the protective genes resulted in an ameliorated NK-mediated lysis and macrophage-mediated phagocytosis in vitro.

With a multiplex method of genome editing, one potential concern was alteration in pluripotency of the edited hPSCs. Here, the authors demonstrated that the engineered hPSCs retained pluripotency-based gene expression and differentiated readily into all 3 germ layers. Off-target events of genome editing were also examined and detected at 3 sites including 1 pseudogene HLA-H and 2 intronic positions. There was also a 95-kb deletion containing MIR6891 and 4 pseudogenes. However, none of these events affected the pluripotency and differentiation capacity of the engineered hPSCs. Thus, important questions that are of immediate interest include: (1) what signals are driving the residual T-cell response towards the engineered hPSCs and can these be further inhibited by genome editing or other measures and (2), beyond the 3 germ layers, do cellular end-products differentiated from such engineered hPSCs retain normal characteristics and desired functions?

This report took the first steps to illustrate the possibility of using genome editing technology to generate hPSCs that have the potential to overcome both adaptive and innate immune barriers and is thus highly useful to the field of cell therapy.

1. de Rham C, Villard J. Potential and limitation of HLA-based banking of human pluripotent stem cells for cell therapy. J Immunol Res. 2014; 2014:518135

2. Han X, Wang M, Duan S, et al. Generation of hypoimmunogenic human pluripotent stem cells. Proc Natl Acad Sci U S A. 2019; 116:10441–10446

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A Cellular Taxonomy of the Bone Marrow Stroma in Homeostasis and Leukemia

Baryawno N, Przybylski D, Kowalczyk MS, et al. Cell. June 2019

In the bone marrow, nonhematopoietic “niche” cells play an important role in forming stem cell niches that influence hematopoietic stem cell (HSC) differentiation and function.1 Several populations of niche cells have been identified, including mesenchymal stem cells (MSCs), bone marrow-derived endothelial cells (BMECs), and osteolineage cells (OLCs). However, a comprehensive landscape of these cells is missing, limiting our ability in targeting them therapeutically for treating various diseases.

In this study, Baryawno et al2 employed single-cell RNA sequencing (scRNA-seq) to study isolated bone marrow niche cells from C57BL/6 mice, and based on their gene expressions charted a complete census of murine bone marrow non-HSCs. They identified 17 non-HSC cell subsets that can be clustered into 6 distinct cell populations including MSCs, BMECs, OLCs, and other cell types including chondrocytes, fibroblasts, and pericytes. Contrasting the conventional notion that all OLCs differentiate from MSCs, diffusion trajectory analysis identified a distinct differentiation trajectory from MSCs to one but not the other OLC subsets, supporting that the 2 OLC subsets have distinct differentiation origins. Other important information uncovered by their study included: (1) 1 of the 5 fibroblast subsets was identified to express Cxcl12, indicating their role in facilitating aggressive cancer growth and bone metastases. (2) BMECs can be subdivided into 3 distinct subsets, with the arterial BMECs being the subset that expressed the most abundant “niche factors” regulating HSC function.

This study has thus created a comprehensive cellular taxonomy of normal mouse non-HSC bone marrow cells mapping their roles in supporting HSC differentiation and function. To apply this information to a hematologic disease, the authors studied the effect of acute myeloid leukemia on bone marrow niche cells. Using scRNA-seq, the authors observed that acute myeloid leukemia compromised MSC and OLC development, altered bone formation and breakdown, and modified “niche factor” gene expressions that collectively reduced normal hematopoiesis.

In support, another recent study3 demonstrated that ablative chemotherapy resulted in a significant transcriptional remodeling in niche cells and that expression of Notch delta-like ligand Dll4 by BMECs was critical for lymphoid lineage commitment.

In considering the implications of these findings for transplantation, it is known that bone marrow is a critical site of differentiation of immune cells implicated in alloimmune responses; for instance, B cells implicated antibody-mediated rejection. Bone marrow is also a target site of several experimental tolerance induction strategies involving bone marrow chimerism. Data available through these studies will now serve as a useful atlas for future studies in transplant recipients examining (1) the effect of clinically implemented immunosuppression on bone marrow niche cells, with particular impact on the differentiation of innate and adaptive immune cells and (2) the effect of bone marrow chimerism-based and nonchimerism-based experimental tolerance regimens on bone marrow niche cells.

Information from such studies may open the door for niche cell-targeting therapies inhibiting transplant rejection and possibly promoting transplant tolerance.

1. Wei Q, Frenette PS. Niches for hematopoietic stem cells and their progeny. Immunity. 2018; 48:632–648

2. Baryawno N, Przybylski D, Kowalczyk MS, et al. A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia. Cell. 2019; 177:1915–1932.e16

3. Tikhonova AN, Dolgalev I, Hu H, et al. The bone marrow microenvironment at single-cell resolution. Nature. 2019; 569:222–228

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