Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease

Abstract

Autoimmune diseases (AIDs), a heterogeneous group of immune-mediated disorders, are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs, the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However, stem cell transplantation therapy has limitations, including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology, which allows the generation of patient-specific pluripotent stem cells, could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally, all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS, SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs, including AS, SS and SLE.

Interleukin-2 induces the in vitro maturation of human pluripotent stem cell-derived intestinal organoids

“…hiPSC lysates were prepared using 1× Taq buffer (Takara, Kyoto, Japan) and proteinase K at 55 °C for 3 h and were used for qPCR analysis as described previously 51 . A known concentration of the pCXLE-hFbx15-cont2 plasmid was used to create a standard curve.…”

Comparative analysis of human embryonic stem cell‑derived neural stem cells as an in vitro human model

“…no. WA09; WiCell Research Institute, Madison, WI, USA) were maintained on Matrigel (BD Biosciences, San Diego, CA, USA) in mTeSR1 (StemCell Technologies, Vancouver, BC, Canada) as previously described ( 14 , 15 ).…”

“…no. WA09; WiCell Research Institute) were maintained on Matrigel (BD Biosciences) in mTeSR1 (StemCell Technologies) as previously described ( 14 ). Human embryoid bodies (hEBs) were generated by culturing hESCs in hEB medium consisting of knockout DMEM supplemented with 10% knockout serum replacement, 1% non-essential amino acids, 1 mM L-glutamine (all from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 0.1 mM β-mercaptoethanol (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) on non-coated Petri dishes.…”

Episomal Induced Pluripotent Stem Cells: Functional and Potential Therapeutic Applications

“…Subsequently, episome-free iPSCs can be easily harvested. Although plasmids appear in the first few cell passages immediately after transfection, many studies have used PCR analysis to show complete loss of plasmids and transgenes during extended cell culture 20,[39][40][41][42][46][47][48][49][50][51][52][53][54][55][56][57][58] . The time point of successful loss of episomes varies between different somatic cell types.…”

“…The human cells reported to have been successfully reprogrammed into EiPSCs include fibroblasts, epithelial cells, keratinocytes, mononuclear cells from adult peripheral blood, cord blood cells, amniotic fluid stem cells, mesenchymal stromal cells, lymphoblasts, lamina propria progenitor cells from oral mucosa, and urothelial cells obtained from urine. A summary of these reported human EiPSC sources is shown in Table 1 8,20,21,[32][33][34][35][36][37][38][39][40][41][42][43][47][48][49][50][51][52]55,56,58, . The variety of cell lineages that can be reprogrammed by episomal techniques demonstrates the versatility of this technique, which has been used in many laboratories around the world.…”