Int J Stem Cells.  2024 Feb;17(1):59-69. 10.15283/ijsc23037.

Expression of Major Histocompatibility Complex during Neuronal Differentiation of Somatic Cell Nuclear Transfer-Human Embryonic Stem Cells

• Lee JS
• Lee JE
• Yu SH
• Chun T
• Chang MY
• Lee DR
• Park CH

• KMID: 2552018
• DOI: http://doi.org/10.15283/ijsc23037

Abstract

Human pluripotent stem cells (hPSCs) such as human embryonic stem cells (hESCs), induced pluripotent stem cells, and somatic cell nuclear transfer (SCNT)-hESCs can permanently self-renew while maintaining their capacity to differentiate into any type of somatic cells, thereby serving as an important cell source for cell therapy. However, there are persistent challenges in the application of hPSCs in clinical trials, where one of the most significant is graft rejection by the patient immune system in response to human leukocyte antigen (HLA) mismatch when transplants are obtained from an allogeneic (non-self) cell source. Homozygous SCNT-hESCs (homo-SCNT-hESCs) were used to simplify the clinical application and to reduce HLA mismatch. Here, we present a xeno-free protocol that confirms the efficient generation of neural precursor cells in hPSCs and also the differentiation of dopaminergic neurons. Additionally, there was no difference when comparing the HLA expression patterns of hESC, homo-SCNT-hESCs and hetero-SCNT-hESCs. We propose that there are no differences in the differentiation capacity and HLA expression among hPSCs that can be cultured in vitro. Thus, it is expected that homo-SCNT-hESCs will possess a wider range of applications when transplanted with neural precursor cells in the context of clinical trials.

Keyword

HLA expression; Neuronal differentiation; Human SCNT ESC; Dopaminergic differentiation

Figure

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  Fig. 1 Pluripotent marker expressions of human pluripotent stem cells (hPSCs). (A) alkaline phosphatases staining is the result of using conventional dyes as substrates for measuring the activity of elevated alkaline phosphatase in hPSCs. The characteristics of the fluorescently labeled cells are observed in the general hPSCs morphologies. (B) Reverse transcription polymerase chain reaction analysis reveals the expression of three germ layer markers (PAX6, LMO2, GSC) in embryoid bodies. Scale bar=500 μm. hESC: human embryonic stem cell, hiPSC: human induced pluripotent stem cell, Homo-SCNT-hESC: homozygous somatic cell nuclear transfer-hESC.

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  Fig. 2 Generation of neural precu-rsor cells (NPCs) from human pluripotent stem cells (hPSCs). (A) Sche-matic describing the differentiation protocol of NPCs via spherical neural mass (SNM) from hPSCs. hPSCs transitioned through stages of embryoid body (EB), neural rosette, SNMs, and NPCs. (B-E) Typical morphology of the hPSC colony. (F-I) hPSCs were indu-ced for 5 days with suspended EB. (J-M) After EB attachment, neural rosettes were induced for 5∼10 days. (N-Q) After the selection of the SNMs, they were passaged 3 times for purification. (R-U) After 3∼4 passages, the SNMs were dissociated into single cells. Scale bar=100 μm. hESC: human embryonic stem cell, hiPSC: human indu-ced pluripotent stem cell, Homo-SCNT-hESC: homozygous somatic cell nuclear transfer-hESC.

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  Fig. 3 Neural precursor cell specifications are similarly induced in various human pluripotent stem cells (hPSCs). (A) Neural rosettes were ide-ntified according to rosette markers such as PAX6 and ZO-1. (B) Repre-sentative images indicating the spherical neural mass markers NESTIN and SOX2. (C) After single cell dissocia-tion of neural precursor cells, the left panel also reveals the neural precu-rsor cell markers NESTIN and SOX2. The right panel presents the quantified data from the left panel. Scale bar=50 μm. hESC: human embryonic stem cell, hiPSC: human induced pluripotent stem cell, Homo-SCNT-hESC: homozygous somatic cell nuclear transfer-hESC.

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  Fig. 4 In vitro differentiation potentials of human pluripotent stem cells (hPSCs)-derived neural precursor cells are similar in various hPSCs. (A) Dif-ferentiation states into typical neurons, astrocytes, oligodendrocytes were indicated by the expression markers TUJ1, GFAP, and OLIG2, respectively. (B) Representative images and quantification of individually induced marker positive cells after differentiation for 14 days. Scale bar=50 μm. hESC: human embryonic stem cell, hiPSC: human induced pluripotent stem cell, Homo-SCNT-hESC: homozygous somatic cell nuclear transfer-hESC.

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  Fig. 5 Differentiation of human pluripotent stem cells-neural precursor cells (hPSCs-NPCs) into dopaminer-gic (DA) neurons. (A) Schematic procedures for the production of DA neurons. (B) Approximately one month after hPSCs-NPCs differentiation, TUJ1- and TH- positive neurons were expressed by immunocytochemical cha-racterization. (C) Quantification of the stereological count of TH-positive and TUJ1-positive cells. Scale bar=50 μm. hESC: human embryonic stem cell, hiPSC: human indu-ced pluripotent stem cell, homo-SCNT-hESC: homozygous somatic cell nuclear transfer-hESC.

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  Fig. 6 Major histocompatibility complex (MHC) Class I and II expression increase as neuronal differentiation progresses. MHC Class I and II expression was not detected in undi-fferentiated human pluripotent stem cells. However, MHC Class I and II expression was induced during embryoid body (EB) formation and maintained until the completion of neuronal differentiation. And there were no differences in MHC Class I and II expression patterns between human embryonic stem cell (hESC) and somatic cell nuclear transfer (SCNT)-hESC. Scale bars=200 μm. Homo: homozygous, SNM: spherical neural mass.

Reference

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