The Universal 3D3 Antibody of Human PODXL Is Pluripotent Cytotoxic, and Identifies a Residual Population After Extended Differentiation of Pluripotent Stem Cells

Abstract

Podocalyxin-like protein (PODXL) is a member of CD34 family proteins. It is the protein that carries many post-translational epitopes responsible for various pluripotent surface markers including TRA-1-60, TRA-1-81, GCTM2, GP200, and mAb84. However, PODXL has not attracted the attention of stem cell biologists. Here, we report several features of PODXL mRNA and protein in pluripotent stem cells. Similar to the modificationdependent pluripotent epitopes, PODXL transcripts and carrier protein are also features of pluripotency. PODXL is highly expressed in early human embryos from oocytes up to four-cell stages. During reprogramming of human cells to pluripotency, in contrast to TRA-1-60 and TRA-1-81, PODXL is activated by KLF4 at a very early time of reprogramming. Although TRA-1-60 and TRA-1-81 are completely lost upon differentiation, a residual PODXL + population exists even after extended differentiation and they were identified by the universal human PODXL epitope 3D3. Unlike TRA-1-60 and TRA-1-81 epitopes that are unique to primate pluripotent stem cells (PSCs), PODXL carrier protein can be used as a murine surface marker. Most importantly, antibody to 3D3 epitope causes massive necrosis and apoptosis of human PSCs (hPSCs). We suggest that 3D3 antibody could be employed to eliminate the tumorigenic pluripotent cells in hPSC-derived cells for cell transplantation.

Reprogramming by De-bookmarking the Somatic Transcriptional Program through Targeting of BET Bromodomains

“…BETiPSCs did not express GFP ( Figure 2B), indicating silencing of the transgenes. BETiPSCs activated the pluripotent transcription factors OCT4, NANOG, and SOX2 and expressed the pluripotent surface markers TRA-1-60 and TRA-1-81 ( Figure 2A) (Kang et al, 2016). The expression of OCT4 was not from the transgenic OCT4, because the multiple GFPs co-expressed with OSK were efficiently silenced ( Figure 2B).…”

Anti-podocalyxin antibody exerts antitumor effects via antibody-dependent cellular cytotoxicity in mouse xenograft models of oral squamous cell carcinoma

“…Despite the development of anti-PODXL monoclonal antibodies (mAbs) [ 32 , 33 ], the efficacy of these treatments against oral cancers remains to be fully elucidated. We previously immunized mice with recombinant PODXL, which was purified from the culture supernatant of LN229/ectodomain-PODXL cells [ 17 ].…”

Identification of LEA, a podocalyxin‐like glycoprotein, as a predictor for the progression of colorectal cancer

“…To verify the application potential of LEA, a PODXL recognized by mAb ND‐1 against its sialic acid residues, we comparatively analyzed the LEA/PODXL expression using 3D3 against PODXL and ND‐1. 3D3 is a widely used monoclonal antibody binding to amino acid residues 254‐415 of extracellular domain of the PODXL . Among five CRC cell lines detected, four cell lines could be detected by 3D3 and three‐ones did by ND‐1 (Figure ).…”

“…3D3 is a widely used monoclonal antibody binding to amino acid residues 254-415 of extracellular domain of the PODXL. 31,47 Among five CRC cell lines detected, four cell F I G U R E 6 Characterization of exosomes derived from CL187 cells and CRC patient plasma. A, Representative electron microscope images of exosomes isolated from CL187 cells.…”