To explore the relationship between epidermal stem cells and the developing process of sweat gland in human fetal skin, so as to obtain a hint for future induction of epidermal stem cells to differentiate into sweat gland cells.

RNA interference phenomenon in three different murine ES cell lines (MESPU13, B3, and R1) is reported. A vector(pdsGFP) was used that transcribed hairpin double-stranded RNA of GFP gene to transfect ES cells by using lipofectin. The transient transcription of dsRNA induced RNAi (RNA interference) in the ES cells. That is, the double-stranded RNA of GFP gene potently turned down the expression of the GFP gene. On the hand, the linearized plasmid pdsGFP-puro was electroporated into MESPU13 ES cells, and the expression level of GFP after puromycin screening was turned down obviously in about 30% ES cell clones; and in a few clones, the expression level of GFP was not observed under the fluorescence microscope and GFP mRNA was not detectable by RT-PCR. Further more, another vector (pdsOCT4) was constructed that transcribed double-stranded RNA of OCT-4 gene which is specifically expressed in ES cells. ES cell clones that stably integrated the vector were screened after the electrotransfection of the cells with the above construct. 51 random-selected clones were amplified and 48 of them were checked by semi-quantitative RT-PCR. In 11 of them the mRNA of OCT-4 was undetectable by RT-PCR. This means that RNAi can be used to study mammal and human gene's function in ES cell lines from different strain mice.

To gain an understanding of the expression of beta-NGF in myoblasts transfected by PSVCEP NGF-CAT and the transfecting efficiency of Lipofect AMINE.

To study the effect of primary brain-stem injury on the expression of basic fibroblast growth factor (bFGF) in the reticular formation of medulla oblongata.

The advancement of the study in embryonic stem cells and tissue engineering makes it promising to effectively solve the intractable problems in the present ophthalmic practice. The problems include: no effective measures to regain the visual function of the patients with late stage glaucoma or retinal diseases, shortage of appropriate biocompatible donor tissues in treating ocular surface diseases.

The viability of limbal stem cells with cryopreservation and the treatment effect with cryopreserved limbal autograft transplantation for ocular surface disorders (OSDs) of stem cells deficiency in rabbits.

To investigate the possibility of commitment differentiation of embryonic stem cells (ES cells) induced by corneal limbal stroma.

Using nested polymerase chain reaction (PCR) to perform preimplantation gender diagnosis.

Human immunodeficiency virus type-I (HIV-I), one kind of lentiviruses, was characterized by a complex genome that encodes two regulatory proteins and four accessory proteins in addition to the common gag, pol and env gene products. So far, a few of different types of replication-defective vectors were constructed, the highest viral titer from one of which was above 10(7) TU/ml. Several studies on packaging cell line found that eliminating the four accessory genes would have few effect on transduction ability of vector and split-genome package system could reduce the possibility of producing replication-competent virus. There are two kinds of characters on HIV-I vectors. Firstly, it was highly efficient in transducing CD34(+) human hematopoietic stem/progenitor cells; secondly, repeated injections of the HIV-I vector into animal did not elicit the rejection response. HIV-I vector had an extensive host range.

Besides hematopoietic stem cells, bone marrow also contains another type of stem cells called mesenchymal stem cells (MSCs). With different induced conditions, MSCs have the ability to differentiate into a variety of nonhematopoietic tissue cells, including osteoblasts, chondroblast, adipocytes, myoblasts, astrocytes, and so on. MSCs can be readily obtained from bone marrow by their adhesion to plastic and expansion in culture. Also they can be genetically engineered by transduced target genes. MSCs may be the farget cells for both cell therapy and gene therapy for diseases derived from many different nonhematopoietic tissues.

To evaluate the hematopoietic potentiality and the migration and homing routine of separated as well as cryopreserved umbilical cord blood hematopoietic cells, the BALB/cnu(+) mice were used to establish a murine model. This can prepare for the clinical transplantation and the establishment of a large-scale cord blood bank. The result indicated that the hydroxyethyl starch (HES) sedimentation and DMSO step-by-step cryopreservation procedure resulted in only less losses of hematopoietic progenitor cells and also unharmful to the hematopietic potentiality. We can found evidence for successful transplantation in each mouse which received (1.0 - 2.0) x 10(7) separated or cryopresered hematopoietic cells from cord blood, which lasted for about fifty days. The results demonstrated that (1) HES sedimentation and DMSO cryopreservation procedure can keep the hematopoietic potentiality of cord blood, and so can be used to clinical transplantation or establishment of a cord blood bank; (2) Rich hematopoietic stem cells in human cord blood can cross the xenogenetic barriers and successfully engraft mice; (3) The hematopoietic cells migrated among bone marrow, liver, spleen, lung and kidney in the mice and homed to bone marrow by the end. Cryopreservation may influence the adhesion molecule on the hematopoietic cells and the homing behaviour, but not influence their hematopoietic potentiality.

More works documented recently indicated that human CD34(-) cells exist and are likely to be the precursors of the CD34(+) cells. Anyhow, the CD34(+) enriched populations have already been proved to show long-term reconstitution of hematopoiesis in animals and patients worldwidely. It still remains uncertain whether cells lack of CD34 and Lineage markers are the very best stem cells or maybe the residual embryonic stem cells keeping quiescent in the adult tissues are capable of transfer into hematopoietic stem cells when activated. Since just a negative selection technique is used to collect the CD34(-) cells everywhere for the time being, no final conclusion is convincing about the characterization of CD34(-) cells till a highly purified cell population of CD34(-)/Lin(-) is available. Clinical analysis shows that the most critical factor predicting the stem cell engraftment is the number of the cells infused. The number of nucleated cells in umbilical cord blood to be infused and required to obtain a successful engraftment is superior to 3.7 x 10(7)/kg. However, large dose of T-cell-depleted and purified CD34(+) cells as more than 5 x 10(6)/kg or even a 'megadose' of CD34(+) cells of 10(7)/kg is recommended for allotransplant of mobilized peripheral blood to achieve a high rate of successful engraftment. The delayed engraftment and the relapse of malignancy after cord blood transplantation are major problems. However, CBT is still the best choice of stem cell transplant for the baby patients with non-malignancies. At present, the HLA typing for class I antigens is still achieved with serology in most laboratories. As HLA typing is increasingly defined to higher degree of resolution by DNA probes, it is recommended to check with genotyping when there is a 'match' by the serological phenotyping especially for the unrelated donor/recipient couples. Improvements in DNA-based methods for the detection of numerous HLA alleles have provided the opportunity to investigate the relationship between HLA disparity and transplant complications. About 80% (or even more) of patients in China who might benefit from stem cell transplantation still fail to find suitable donors. It is worthy to adopt the unrelated donors matched or mismatched for those high-risk acute leukemia patients who do not have related matched donors but urgently need transplant. The advanced experience of unrelated mismatched transplant will no doubt be certain to carry weight and be disseminated in China. A great leap forward of the clinical practice and biological study on hematopoietic stem/progenitor cells is expected in the new century to accept the challenge from the world outside China.

The applications of allogeneic bone marrow transplantation (allo-BMT) are limited by the high risk of relapse with primary malignancy. The newly adaptive immunotherapy with donor lymphocyte infusions (DLI) can induce an effective graft-versus-leukemia (GVL) reaction leading to molecular remission and prolonged disease free survival in the majority of patients with relapse of CML and some other hematologic malignancies. Little is known about the mechanisms and the kinetics of GVL, however, increasing evidences show that in the case of existence of chimerism the mature donor T lymphocytes are activated by the specific leukocyte artigen presented in the surface of host antigen presenting cells (APC), resulting in exclusively elimination of leukocyte. Undesired side-effects are the development of graft-versus-host disease (GVHD) and the occurrence of pancytopenia in some patients. Infusions with selective T lymphocytes and G-CSF-mobilized peripheral blood stem cells (PBSC) are the promising treatment of DLI for more patients.

Stem cells of various tissues including hematopoietic tissue in the body are derived from embryonic stem cells (ESC). There exists intricated gene regulation during ESC development and its differentiation into hematopoietic stem cells (HSC). In embryo hematopoiesis development, there are two kinds of hematopoietic types, primitive hematopoiesis and definitive hematopoiesis. The theory of the yolk sac of primitive hematopoiesis is well accepted, while the initial site of definitive hematopoiesis still exists controversy. In present opinion, there are at least two independent sites associated with definitive hematopoiesis, those are, yolk sac and para-aortic splanchnopleura (PAS)/aorta-gonad mesonephros (AGM). Study on the hematopoiesis and its regulation during embryonic ontogeny will benefit not only to the dicovery of the mechanism of some blood disorders, but as well to gene therapy and HSC engineering.

To study the reconstituion of the T-cell populations after allogeneic hematopoietic stem cell transplantation, the peripheral blood samples obtained at different time points post-transplantation in 21 patients were analyzed using CD5-PE and CD3-FITC with flow cytometry. During the first 3 months after transplantation, CD3(-) CD5(+) T cell subsets, representing early thymocytes, remained below normal level, whereas CD3(+) CD5(+) T cell subsets, representing mature T cells, regenerated to normal level. In 9 patients who developed acute graft-versus-host disease (GVHD), the percentage of CD3(+) CD5(+) T cell subsets was significantly higher than that in patients who did not develop acute GVHD (P < 0.05). These results demonstrated that the early recovery of T cells is mainly due to the expansion of mature T cell populations, and the over-expansion of mature T (CD3(+) CD5(+)) cells is responsible for the development of acute GVHD.

Transfer of drug resistance genes into hematopoietic cells is an attractive approach to protect hematopoietic system from the toxic effects by chemotherapeutic agents in cancer patients. In this study, transduction of mdr-1 in combination with dihydrofolate reductase (dhfr) gene was performed, and the expression of exogenous genes and chemoprotection capacity in mouse bone marrow cells were observed. The results showed that approximately 15% of bone marrow cells transfected with the retroviral vector expressed mdr-1 as assayed by flow cytometry. Gene transfer resulted in about 0.9 - 13 fold and 0.5 - 2.6 fold increase in the resistance of CFU-GM to taxol and methotrexate in vitro, respectively (P < 0.05). Moreover, seven months after transplantation to syngeneic mice with mdr-1 and dhfr-transfected bone marrow cells, peripheral blood cells in recipients were still positive for gp170 as evaluated by FACS as well as for mdr-1 and dhfr by PCR amplification. These results indicate that hematopoietic progenitors can be transfected by retrovirus containing mdr-1 and dhfr genes, and that functional drug resistance accompanies their expressions. Furthermore, genetic chimerism might exist in hematopoietic stem cells. In conclusion, transfer and expression of mdr-1 and dhfr genes in bone marrow cells might be applicable in gene therapy research in cancer patients.

Allogeneic hematopoietic stem cell transplantation is curative therapy for many malignant hematologic and genetic disorders. The pathways by which T cell are regenerated in vivo are of central importance for host immune competence after the transplantation. Thymic-dependent and thymic-independent pathways of T-cell regeneration are primarily determined by thymus function, and that thymic-independent pathways are generally inadequate for restoration of host immunocompetence. T cell regeneration of adult recipients is largely through thymic-independent pathways due to reduced thymic regeneration capacity, resulting in quantitative defficiencies in T-cell number and severe restriction in the diversity of the regenerated T-cell receptor (TCR) repertoire. New strategies to enhance thymic function in man after BMT would hold great therapeutic potential. Activation of donor T cells is the cause of GVHD, the induction of anergy can inactivate specific sets of alloreactive T cells in the donor stem cells, which can prevent GVHD without inhibiting the entire T-cell repertoire.

The enumeration of CD34(+) cells by flow cytometry is commonly employed to assess hematopoietic stem/progenitor cells in cord blood, peripheral blood and apheresis products. Interlaboratory variation of CD34(+) cells enumeration is exceedingly large. Factors contributing to those variation, currently established flow cytometry protocols and comparison between these protocols were reviewed. CD34(+) cells in 45 cord blood samples, 12 normal bone marrows and 4 apheresis products were also enumerated in our laboratory by using ProCUNT kit, and results showed that the ProCUNT is highly standardizaed and could assist in reducing interlaboratory variation of CD34(+) cells.

In order to explore the expansive effect of human placental cell-free suspension (HPCFS) on bone marrow hematopoietic stem/progenitor cells, and to compare the effect of HPFCS with some cytokines and their combination, human marrow CFU-GM, CFU-GEMM and BFU-E were assayed in a semisolid methyl cellulose culture system using HPCFS, IL-3, GM-CSF, and IL-3 + IL-6 + GM-CSF + EPO as colony stimulating factors, respectively. The results showed that HPCFS stimulated the growth of CFU-GM, CFU-GEMM and BFU-E and the optimal concentrations for stimulating effect were 200 - 300 micro g protein/L, and the yield of 3 kinds of colony in HPCFS group was higher than that in IL-3, GM-CSF and IL-3 + IL-6 + GM-CSF + EPO groups. The expansive effect of HPCFS on marrow progenitors was superior to IL-3, GM-CSF and IL-3 + IL-6 + GM-CSF + EPO. Human placental cell-free suspension contained a variety of cytokines to stimulate proliferation of hematopoietic stem/progenitor cells, and it could be used as an efficacious and inexpensive agent to expand hematopoietic stem/progenitor cells in vitro.