• Development and Reproduction
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• v.23 no.2
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• pp.79-92
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• 2019

Humanized mice, containing engrafted human cells and tissues, are emerging as an important in vivo platform for studying human diseases. Since the development of Nod scid gamma (NSG) mice bearing mutations in the IL-2 receptor gamma chain, many investigators have used NSG mice engrafted with human hematopoietic stem cells (HSCs) to generate functional human immune systems in vivo, results in high efficacy of human cell engraftment. The development of NSG mice has allowed significant advances to be made in studies on several human diseases, including cancer and graft-versus-host-disease (GVHD), and in regenerative medicine. Based on the human HSC transplantation, organ transplantation including thymus and liver in the renal capsule has been performed. Also, immune reconstruction of cells, of the lymphoid as well as myeloid lineages, has been partly accomplished. However, crosstalk between pluripotent stem cell derived therapeutic cells with human leukocyte antigen (HLA) mis/matched types and immune CD3 T cells have not been fully addressed. To overcome this hurdle, human major histocompatibility complex (MHC) molecules, not mouse MHC molecules, are required to generate functional T cells in a humanized mouse model. Here, we briefly summarize characteristics of the humanized mouse model, focusing on development of CD3 T cells with MHC molecules. We also highlight the necessity of the humanized mouse model for the treatment of various human diseases.

• The Korea Journal of Herbology
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• v.28 no.4
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• pp.7-16
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• 2013

Objectives : Brassica campestris var narinosa (BCN), Canavalia gladiata DC semen (CGD) and their combinational prescription (BCN+CGD) have been use to demonstrate to regulate hematopoiesis. In the current study, we investigated whether Brassica campestris var narinosa, Canavalia gladiata DC semen and their combinational prescription is related to hemato-potentiating function using Sca-$1^+$ hematopoietic stem cells (Sca-$1^+HSCs$) as a testing system. Methods : Sca-$1^+HSCs$ isolated from femur in C57bl/6 mice with leukopenia and thrombocytopenia induced by cyclophosphamide (CTX). Then, Real-time PCR was performed to measure the mRNA expression, ELISA and haematopoiesis-related gene (EPO, TPO, IL-3, SCF, c-kit, GM-CSF), the phosphorylation of JAK2, GATA-1 and STAT-5a/b were observed by western blot, and the numbers of $CD117^+/Sca-1^+$ cell and the number of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E), semisolid clonogenic assay was performed. Result : When Sca-$1^+HSCs$ were treated with Brassica campestris var narinosa, Canavalia gladiata DC semen and their combinational prescription with rIL-3/rSCF, the expression of haematopoiesis-related (EPO, TPO, IL-3, SCF, c-kit, and GM-CSF) were significantly increased at the levels of mRNA as well as production in Sca-$1^+HSCs$. Additionally, CGS enhanced phosphorylation of JAK2, GATA-1, and signal transducer and activator of transcription-5a/b (STAT-5a/b) in Sca-$1^+HSCs$. Furthermore, their combinational prescription (BCN+CGD) significantly enhanced the growth rate of granulocyte erythrocyte monocyte macrophage colony-forming units (CFU-GEMM) and erythroid burst forming units (BFU-E) in vitro. Conclusion : These result suggest that Brassica campestris var narinosa (BCN) and Canavalia gladiata DC have hematopoietic enhancement via hematopoietic cytokine-mediated JAK2/GATA-1/STAT-5a/b pathway, and their combinational prescription (BCN+CGD) has superior hematopoietic enhancement to those of individual extracts.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.4
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• pp.265-271
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• 2012

HoxB4, a homeodomain-containing transcription factor, is involved in the expansion of hematopoietic stem cells and progenitor cells in vivo and in vitro, and plays a key role in regulating the balance between hematopoietic stem cell renewal and cell differentiation. However, the biological activity of HoxB4 in other cells has not been reported. In this study, we investigated the effect of overexpressed HoxB4 on cell survival under various conditions that induce death, using the Ba/F3 cell line. Analysis of phenotypical characteristics showed that HoxB4 overexpression in Ba/F3 cells reduced cell size, death, and proliferation rate. Moreover, the progression from early to late apoptotic stages was inhibited in Ba/F3 cells subjected to HoxB4 overexpression under removal of interleukin-3-mediated signal, leading to the induction of cell cycle arrest at the G2/M phase and attenuated cell death by Fas protein stimulation in vitro. Furthermore, apoptotic cell death induced by doxorubicin-treated G2/M phase cell-cycle arrest also decreased with HoxB4 overexpression in Ba/F3 cells. From these data, we suggest that HoxB4 may play an important role in the regulation of pro-B cell survival under various apoptotic death environments.

• KSBB Journal
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• v.14 no.5
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• pp.593-599
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• 1999

In this study, optimal conditions to infect CD34 positive cells containing hematopoietic stem cells obtained from cord blood and bone marrow were found using two different retroviral vectors expressing human growth hormone (hGH) and $\beta$-galactosidase. CD34 positive cells were successfully infected with recombinant retroviruses only when the CD34 positive cells were co-cultured with packaging cells secreting recombinant retroviruses. To find the highest infection efficiency for the gene transfer, CD34 positive cells from cord blood were co-cultured with packaging cells secreting recombinant retroviruses encoding E. coli lacZ gene. The highest infection efficiency was obtained when CD34 positive cells were cultured for 3 days, and then co-culturing was done for another 2 days. When CD34 positive cells from bone marrow were co-cultured with packaging cells secreting recombinant retroviruses encoding hGH gene, the maximum amount of hGH was also secreted at the same conditions found above, i.e. 3 days of culture and 2 days of co-culture. These results show that there are optimal conditions for the gene transfer into hematopoietic stem cells regardless of sources of target cells or retroviral vectors used to infect.

• Journal of Periodontal and Implant Science
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• v.51 no.5
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• pp.329-341
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• 2021

Purpose: Periodontal treatment aims at complete regeneration of the periodontium, and developing strategies for periodontal regeneration requires a deep understanding of the tissues composing the periodontium. In the present study, the stemness characteristics and gene expression profiles of cementum-derived cells (CDCs) were investigated and compared with previously established human stem cells. Candidate marker proteins for CDCs were also explored. Methods: Periodontal ligament stem cells (PDLSCs), pulp stem cells (PULPSCs), and CDCs were isolated and cultured from extracted human mandibular third molars. Human bone marrow stem cells (BMSCs) were used as a positive control. To identify the stemness of CDCs, cell differentiation (osteogenic, adipogenic, and chondrogenic) and surface antigens were evaluated through flow cytometry. The expression of cementum protein 1 (CEMP1) and cementum attachment protein (CAP) was investigated to explore marker proteins for CDCs through reverse-transcription polymerase chain reaction. To compare the gene expression profiles of the 4 cell types, mRNA and miRNA microarray analysis of 10 samples of BMSCs (n=1), PDLSCs (n=3), PULPSCs (n=3), and CDCs (n=3) were performed. Results: The expression of mesenchymal stem cell markers with a concomitant absence of hematopoietic markers was observed in PDLSCs, PULPSCs, CDCs and BMSCs. All 4 cell populations also showed differentiation into osteogenic, adipogenic, and chondrogenic lineages. CEMP1 was strongly expressed in CDCs, while it was weakly detected in the other 3 cell populations. Meanwhile, CAP was not found in any of the 4 cell populations. The mRNA and miRNA microarray analysis showed that 14 mRNA genes and 4 miRNA genes were differentially expressed in CDCs vs. PDLSCs and PULPSCs. Conclusions: Within the limitations of the study, CDCs seem to have stemness and preferentially express CEMP1. Moreover, there were several up- or down-regulated genes in CDCs vs. PDLSCs, PULPSCs, and BMSCs and these genes could be candidate marker proteins of CDCs.

• Proceedings of the Membrane Society of Korea Conference
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• 2004.05a
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• pp.55-58
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• 2004

Cell separation from peripheral blood was investigated using surface-modified polyurethane (PU) membranes with different functional groups. Both red blood cells and platelets could pass through unmodified PU and PU-SO$_3$H membranes, while the red blood cells preferentially passed through PU-N(C$_2$H$_{5}$ )$_2$ and PU-NHC$_2$H$_4$OH membranes. The permeation ratio of T and B cells was less than 25% for the surface-modified and unmodified PU membranes. CD34$^{+}$ cells have been recognized as various kinds of stem cells including hematopoietic and mesenchymal stem cells. The adhesiveness of CD34$^{+}$ cells on the PU membranes was found to be higher than that of red blood cells, platelets, T cells or B cells. Overall, the adhesiveness of blood cells on the PU membranes increased in the following order: red blood cells $\leq$ platelets ＜ T cells $\leq$ B cells ＜ CD34$^{+}$ cells. Treatment of PU-COOH membranes with a human albumin solution to detach adhered blood cells, allowed recovery of mainly CD34$^{+}$ cells in the permeate, while both red blood cells and platelets could be isolated in the permeate using unmodified PU membranes. The PU membranes showed different permeation and recovery ratios of specific cells depending on the functional groups attached to the membranes.mbranes.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.3
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• pp.167-174
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• 2012

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-$kit^+$ bone marrow cells (c-$kit^+$ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-$kit^+$ BM cells that give rise to $CD2^+CD8^+$ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-$kit^+$ BM cells than those of controls. And, we compared the ability of the cytotoxicity of $CD2^+CD8^+$ NK cells differentiated by cytokines from c-$kit^+$ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100 : 1 for 4 h once a week. In results, $CD2^+CD8^+$ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-$kit^+$ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-$kit^+$ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

• IMMUNE NETWORK
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• v.23 no.2
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• pp.17.1-17.16
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• 2023

Latent membrane protein 2A (LMP2A), a latent Ag commonly expressed in Epstein-Barr virus (EBV)-infected host cells, is a target for adoptive T cell therapy in EBV-associated malignancies. To define whether individual human leukocyte antigen (HLA) allotypes are used preferentially in EBV-specific T lymphocyte responses, LMP2A-specific CD8⁺ and CD4⁺ T cell responses in 50 healthy donors were analyzed by ELISPOT assay using artificial Ag-presenting cells expressing a single allotype. CD8⁺ T cell responses were significantly higher than CD4⁺ T cell responses. CD8⁺ T cell responses were ranked from highest to lowest in the order HLA-A, HLA-B, and HLA-C loci, and CD4⁺ T cell responses were ranked in the order HLA-DR, HLA-DP, and HLA-DQ loci. Among the 32 HLA class I and 56 HLA class II allotypes, 6 HLA-A, 7 HLA-B, 5 HLA-C, 10 HLA-DR, 2 HLA-DQ, and 2 HLA-DP allotypes showed T cell responses higher than 50 spot-forming cells (SFCs)/5×105 CD8⁺ or CD4⁺ T cells. Twenty-nine donors (58%) showed a high T cell response to at least one allotype of HLA class I or class II, and 4 donors (8%) had a high response to both HLA class I and class II allotypes. Interestingly, we observed an inverse correlation between the proportion of LMP2A-specific T cell responses and the frequency of HLA class I and II allotypes. These data demonstrate the allele dominance of LMP2A-specific T cell responses among HLA allotypes and their intra-individual dominance in response to only a few allotypes in an individual, which may provide useful information for genetic, pathogenic, and immunotherapeutic approaches to EBV-associated diseases.

• Molecules and Cells
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• v.24 no.3
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• pp.343-350
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• 2007

Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.

• BMB Reports
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• v.48 no.8
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• pp.427-428
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• 2015

Despite high interests on microenvironmental regulation of leukemic cells, little is known for bone marrow (BM) niche in leukemia patients. Our recent study on BMs of acute myeloid leukemia (AML) patients showed that the mesenchymal stromal cells (MSCs) are altered during leukemic conditions in a clinical course-dependent manner. Leukemic blasts caused reprogramming of transcriptomes in MSCs and remodeling of niche cross-talk, selectively suppressing normal primitive hematopoietic cells while supporting leukemogenesis and chemo-resistance. Notably, differences in BM stromal remodeling were correlated to heterogeneity in subsequent clinical courses of AML, i.e., low numbers of mesenchymal progenitors at initial diagnosis were correlated to complete remission for 5-8 years, and high contents of mesenchymal progenitor or MSCs correlated to early or late relapse, respectively. Thus, stromal remodeling by leukemic cell is an intrinsic part of leukemogenesis that can contribute to the clonal dominance of leukemic cells over normal hematopoietic cells, and can serve as a biomarker for prediction of prognosis. [BMB Reports 2015; 48(8): 427-428]