Hematopoietic stem cells (HSCs) are the self‐renewing, multipotent progenitors that give rise to all types of mature blood cells. The hallmark properties of HSCs are the ability to balance self‐renewal versus differentiation cell fate decisions to provide sufficient primitive cells to sustain haematopoiesis, while generating more mature cells with specialized capacities. In the present experiment, we optimized the techniques for isolation and identification of hematopoietic stem cells from cow peripheral blood. The objective of this study was to optimize the more accurate methodology for separation of mononuclear cells (MNCs) from peripheral blood and identification of HSCs by using a specific cell surface marker i.e. CD34. A total 10 peripheral blood samples were collected from Holstein dairy cows from jugular vein. We used Ficoll 400 in different concentrations from 1 to 12% and Ficollpaque Plus (1.077 g/ml) at different centrifugation speed and time. After Giemsa staining, we found more than 98% recovery of monocytes with Ficollpaque Plus (1.077 g/ml). It was demonstrated that Ficollpaque Plus (1.077 g/ml) and centrifugation at 400xg for 30 min is the best method for separation of MNCs from bovine peripheral blood. Separated MNCs were immediately subjected for magnetic activated cell sorting (MACS) by using CD34 microbead kit. HSCs (CD34+ cells) recovery was 0.307% of peripheral blood. Peripheral blood MNCs and CD34+ cells were morphologically characterized by Giemsa staining. CD34+ cells were also confirmed by immunochemistry using FITC conjugated CD34 antibodies. HSCs were also confirmed by progenitor assay including burst forming unit‐erythroid (BFU‐E), colony forming cells‐ granulocyte (CFC‐G), colony forming cells‐ macrophage (CFC‐M), colony forming cells‐ granulocyte macrophage (CFU‐GM) and colony forming cells‐ granulocyte erythroid macrophage monocyte (CFCGEMM) on Methocult 4435.

This study was performed to investigate the effect of extract mixture(IPGE) drink from Inonotus Obliquus, Phellinus Linteus and Ganoderma Lucidum on hematopoietic stem cells and lymphocyte subset[lymphocyte, CD4+ T cell, CD8+ T cell, Natural Killer(NK) cells] of blood in 37 participants who were healthy and about 40~70 years old. They were divided into two groups; extract mixture drink administration group(n=27) and placebo administration group(n=12). They were given the test drink daily for 4 weeks. Blood was obtained from the subjects every two week in the beginning of administration day to evaluate the CD34+ hematopoietic stem cells and immune cells. As results, CD34+ hematopoietic stem cells were significanly increased after taking IPGE drink for 4 weeks compared to that before taking the drink (p < 0.001). There was no significant changes in number of lymphocytes, CD4+ T cells, CD8+ T cells, NK cells and in the ratio of CD4+/CD8+ cell after taking the test drink. From these results, it was suggested that IPGE have a good health effect by promoting the proliferation of the hematopoietic stem cells.

조혈 줄기 세포에의 효과적인 유전자 전달은 유전자 치료의 새로운 가능성을 제시할 수 있다. 레트로바이러스를 이용한 유전자 전달 기술은 많은 기초 연구와 임상 시도가 이루어진 대표적인 바이러스이다. 그러나 현재 사용되고 있는 in vitro에서의 조혈 줄기 세포에의 유전자 도입은 조혈 줄기 세포의 분화 유도, 자기 복제 능력과homing 능력의 저하 등 많은 문제점이 있다. 본 연구는 이러한 문제점을 극복하기 위한 방법으로서 마우스의 대퇴골에 직접 레트로바이러스를 이식하는 IBM (Intra-Bone Marrow) 방법을 이용하여 조혈 줄기 세포에의 효과적인 유전자 도입을 시도하였다. IBM 이식 2주 후 마우스의 각 조직을 분석한 결과, 골수뿐 아니라 림파절, 비장, 간장 세포 등에서 유전자가 안정적으로 발현하는 것을 관찰하였다. 또한, 6.4+-2.7%의 골수조직 존재 조혈줄기/전구세포에서 도입된 유전자가 안정적으로 발현하고 있는 사실을 확인하였다. 본 연구의 결과를 바탕으로 IBM 이식 방법을 이용한 생체 조직 내 레트로바이러스의 유전자 도입은 조혈 줄기 세포를 이용한 유전자 치료에 매우 효과적인 방법이라는 사실을 시사해주고 있다.

Hematopoietic stem cells (HSC) are multipotent cells that reside in the bone marrow and replenish all adult hematopoietic lineages throughoutthe lifetime. In this study, we analyzed the expression of receptors of P2Y10, purinergic receptor families in murine hematopoietic stem cells, hematopoietic progenitor cells. In addition, the biological activity of P2Y10 was investigated with B lymphocyte cell line, Ba/F3 in effect to cell growth and cell cycle. From the analysis of expression in hematopoieticstem cell. and progenitor with RT-PCR, P2Y10 was strongly expressed in murine hematopoieticstem cells (c-kit+ Sca-l+ Lin-) and progenitor cell population, such as c-kit- Sca-l+ Lin-, c-kit+ Sca-l- Lin- and c-kit- Sca-l- Lin-. To investigate the biological effects by P2Y10, retroviral vector from subcloned murine P2Y10 cDNA was used fur gene introduction into Ba/F3 cells, and stable transfectant cells were obtained by flow cytometry sorting. In cell proliferation assay, the proliferation ability of P2Y10 receptor gene­transfected cells was strongly inhibited, and the cell cycle was arrested at G1 phase. These result suggest that the P2Y10 may be involved the biological activity in hematopoietic stem cells and immature B lymphocytes.

Reactive oxygen species(ROS)에 의한 산화적 손상은 냉동보존 과정과 체외 배양과정 중 세포 생존률 감소의 주된 요인 중 하나이며, 특히 줄기세포의 경우 냉동보존 후 쉽게 분화하거나 사멸하는 경향이 있음이 잘 알려져 있다. 따라서 본 연구는 체외 배양된 인간 조혈모 줄기세포의 냉동보존 시 선별된 항산화제를 처리하여 항산화제가 줄기세포의 생존 및 자동분화에 미치는 영향을 조사하고자 하였다. 해동 후 세포의 생존률은 -tocopherol

배아 줄기세포는 세포 치료 목적을 위한 재료로써 매우 큰 잠재력을 가지고 있으며, 이러한 잠재력의 실현을 위해서 세포의 운명에 결정적인 역할을 하는 요소들을 확인하고 특정 세포의 대량 생산을 위한 방법을 개발하여야 한다. 조혈과정은 폭넓게 연구되어 왔으며, 배아 줄기세포로부터 조혈세포의 분화는 lineage commitment에 관한 연구에 좋은 모델이 된다. 본 연구에서는, 두 종류의 마우스 배아 줄기세포주 TC-1과 B6-1를 이용하여 그 특성과 조

Coculture of HSC with bone marrow-derived mesenchymal stem cells (BM-MSCs) is one of used methods to increase cell numbers before transplant to the patients. However, because of difficulties to purify HSCs after coculture with BM-MSCs, it needs to develop a method to overcome the problem. In the present study, we have examined whether a culture insert placed over a feeder layer might support the expansion of HSCs within the insert. cells isolated from the umbilical cord blood by using midiMACS were divided into three groups. A group of 1 cells were grown on a culture insert without feeder layer (Direct). The same number of HSCs was directly cocultured with BM-MSCs (Contact). The third group was placed onto an insert below which BM-MSCs were grown (Insert). To distinguish feeder cells from HSCs, BM-MSCs was pre-labeled fluorescently with PKH26 and 1 cells were seeded in the culture dishes. After culture for 13 days, the expansion factor (x) of HSCs that were grown without feeder layer (Direct) was In contrast, the number of HSCs directly cocultured with feeder layer was 59.6 0.5 and that of HSCs cultured onto an insert was The percentage of BM-MSCs cells remained being fluorescent was after culture. Immune-phenotypically large proportion of cultured cells were founded to be differentiated into myeloid/monocyte progenitor cells. The ability of BM-MSCs, fetal lung, cartilage and brain tissue cells to support ex vivo expansion of HSCs was also examined using the insert. After 11 days of coculture with each of these cells, the expansion factor of HSCs was 15.0, 39.0, 32.0 and 24.0, respectively. Based upon these observations, it is concluded that the coculture method using insert is very effective to support ex vivo expansion of HSCs and to eliminate the contamination of other cells used to coculture wth HSCs.