Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs).

BACKGROUND: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. METHODS: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. RESULTS: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. CONCLUSIONS: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

Prevention against diffuse spinal cord astrocytoma: can the Notch pathway be a novel treatment target?

This study was designed to investigate whether the Notch pathway is involved in the development of diffuse spinal cord astrocytomas. BALB/c nude mice received injections of CD133(+) and CD133(-) cell suspensions prepared using human recurrent diffuse spinal cord astrocytoma tissue through administration into the right parietal lobe. After 7-11 weeks, magnetic resonance imaging was performed weekly. Xenografts were observed on the surfaces of the brains of mice receiving the CD133(+) cell suspension, and Notch-immunopositive expression was observed in the xenografts. By contrast, no xenografts appeared in the identical position on the surfaces of the brains of mice receiving the CD133(-) cell suspension, and Notch-immunopositive expression was hardly detected either. Hematoxylin-eosin staining and immunohistochemical staining revealed xenografts on the convex surfaces of the brains of mice that underwent CD133(+) astrocytoma transplantation. Some sporadic astroglioma cells showed pseudopodium-like structures, which extended into the cerebral white matter. However, it should be emphasized that the subcortex xenograft with Notch-immunopositive expression was found in the fourth mouse received injection of CD133(-) astrocytoma cells. However, these findings suggest that the Notch pathway plays an important role in the formation of astrocytomas, and can be considered a novel treatment target for diffuse spinal cord astrocytoma.

Stem cell signaling as a target for novel drug discovery: recent progress in the WNT and Hedgehog pathways.

One of the most exciting fields in biomedical research over the past few years is stem cell biology, and therapeutic application of stem cells to replace the diseased or damaged tissues is also an active area in development. Although stem cell therapy has a number of technical challenges and regulatory hurdles to overcome, the use of stem cells as tools in drug discovery supported by mature technologies and established regulatory paths is expected to generate more immediate returns. In particular, the targeting of stem cell signaling pathways is opening up a new avenue for drug discovery. Aberrations in these pathways result in various diseases, including cancer, fibrosis and degenerative diseases. A number of drug targets in stem cell signaling pathways have been identified. Among them, WNT and Hedgehog are two most important signaling pathways, which are the focus of this review. A hedgehog pathway inhibitor, vismodegib (Erivedge), has recently been approved by the US FDA for the treatment of skin cancer, while several drug candidates for the WNT pathway are entering clinical trials. We have discovered that the stem cell signaling pathways respond to traditional Chinese medicines. Substances isolated from herbal medicine may act specifically on components of stem cell signaling pathways with high affinities. As many of these events can be explained through molecular interactions, these phenomena suggest that discovery of stem cell-targeting drugs from natural products may prove to be highly successful.

Rapid and efficient reprogramming of human amnion-derived cells into pluripotency by three factors OCT4/SOX2/NANOG.

Reprogramming human somatic cells to pluripotency represents a valuable resource for research aiming at the development of in vitro models for human diseases and regenerative medicines to produce patient-specific induced pluripotent stem (iPS) cells. Seeking appropriate cell resources for higher efficiency and reducing the risk of viral transgene activation, especially oncogene activation, are of significance for iPS cell research. In this study, we tested whether human amnion-derived cells (hADCs) could be rapidly and efficiently reprogrammed into iPS cells by the defined factors: OCT4/SOX2/NANOG. hADCs from normal placenta were isolated and cultured. The 3rd passage cells were infected with the lentiviral vectors for the delivery of OCT4, SOX2, and NANOG. Afterwards, the generated iPSCs were identified by morphology, pluripotency markers, global gene expression profiles, and epigenetic status both in vitro and in vivo. The results showed that we were able to reprogram hADCs by the defined factors (OCT4/SOX2/NANOG). The efficiency was significantly high (about 0.1%), and the typical colonies appeared on the 9th day after infection. They were similar to human embryonic stem (ES) cells in morphology, proliferation, surface markers, gene expression, and the epigenetic status of pluripotent cell-specific genes. Furthermore, these cells were able to differentiate into various cell types of all three germ layers both in vitro and in vivo. These results demonstrate that hADCs were an ideal somatic cell resource for the rapid and efficient generation of iPS cells by OCT4/SOX2/NANOG.

Comparison of three methods for cryopreservation of human embryonic stem cells.

OBJECTIVE: To establish reliable methods for cryopreservation of human embryonic stem cells (hESCs). DESIGN: Prospective experimental study. SETTING: University laboratory. PATIENT(S): One hESC line. INTERVENTION(S): The attachment rates and recovery rates of cryopreserved hESCs using three different cryopreservation methods were compared. MAIN OUTCOME MEASURE(S): The hESCs were frozen and thawed by conventional cryopreservation, programmable cryopreservation, and vitrification method. The efficiency of cryopreservation was assessed by attachment rate and recovery rate. RESULT(S): The attachment rate and recovery rate after thawing of hESCs frozen by the conventional cryopreservation method were significantly lower than those of hESCs frozen by programmed cryopreservation and vitrification methods. Vitrification resulted in the highest attachment rate and recovery rate compared with the other two methods. Human ESCs after vitrification and programmable cryopreservation still expressed pluripotent markers, maintained normal karyotype, and retained their pluripotency. CONCLUSION(S): Our data show that programmable cryopreservation and vitrification methods are appropriate for cryopreservation of hESCs, whereas the conventional slow-rate freezing method is not appropriate for cryopreservation of hESCs.

Pax6 haploinsufficiency causes abnormal metabolic homeostasis by down-regulating glucagon-like peptide 1 in mice.

Heterozygosity for the Pax6 allele is associated with impaired glucose tolerance in humans. With a Pax6 mutant mouse model, we found many of the metabolic abnormalities were consistent with the effects of down-regulating the expression of glucagon-like peptide 1 (GLP-1). In addition to impaired glucose tolerance, adult heterozygous mutant mice (Pax6(m/+)) secreted less insulin responding to glucose and arginine administration compared with control mice. Moreover, Pax6(m/+) mice showed increased food intake compared with control mice, although they were resistant to diet-induced fat accumulation. Indeed, levels of circulating GLP-1 and intestinal transcription of Gcg/Proglucagon were dramatically reduced in Pax6(m/+) mice. Mutated Pax6 also failed to activate the Gcg/Proglucagon promoter by in vitro transfection assay. Finally, administering the GLP-1 receptor agonist exendin-4 to Pax6(m/+) mice largely reversed their abnormal food intake, glycemic excursion, and insulin secretion. Our studies suggested that disruption of metabolic homeostasis mainly caused by Pax6 haploinsufficiency was mainly mediated by down-regulation of GLP-1. Administration of exendin-4 may be a useful therapy in humans with a similar mutation.

[Experimental study on repairing damage of corneal surface by mesenchymal stem cells transplantation].

OBJECTIVE: To investigate the survival, migration and differentiation of the cultured human mesenchymal stem cells derived from bone marrow (hMSCs) after transplanted onto the alkaline burn rabbit cornea. METHODS: Alkaline burn on rabbit corneas was induced with NaOH solution. One month later, hMSCs cultured with a feeding of amniotic membrane were transplanted onto the surface of alkaline burn rabbit corneas. Amniotic membrane alone transplantation was used at the same time as control group. One month after transplantation, the changes of corneal morphology were analyzed by clinical observations and HE staining. Immunohistochemistry was carried out with the antibodies against human nuclei and cytokeratin 12 to investigate the distribution and differentiation of hMSCs. RESULTS: The rabbit cornea became totally opaque one month after alkaline burn. Blood vessels could be seen within the superficial layer and stroma. The surface of cornea was rough and dry. There were many goblet cells found in the corneal epithelial layer. One month after transplantation of hMSCs, the surface of alkaline burn cornea became smoother. The amount of the new blood vessels of the cornea reduced and the goblet cells disappeared. Anti-human nuclei antibody staining showed positive cells on the surface layer and superficial stroma while cytokeratin 12 positive cells were only present in the epithelium layer. In the amniotic membrane transplantation control group, no clinical improvement of the cornea was found. Goblet cells were still seen on the corneal surface. Both anti-human nuclei antibody and cytokeratin 12 staining were negative. CONCLUSIONS: hMSCs survive and migrate to stroma after transplanted onto the surface of alkaline burn rabbit cornea. No rejection is detected. hMSCs on the corneal surface differentiate to corneal epithelium, where those migrated to the stroma differentiate to cells other than epithelium. hMSCs transplantation decrease corneal conjunctivization after alkaline burn.

Effect of human neural progenitor cells on injured spinal cord.

OBJECTIVE: To study whether human neural progenitor cells can differentiate into neural cells in vivo and improve the recovery of injured spinal cord in rats. METHODS: Human neural progenitor cells were transplanted into the injured spinal cord and the functional recovery of the rats with spinal cord contusion injury was evaluated with Basso-Beattie-Bresnahan (BBB) locomotor scale and motor evoked potentials. Additionally, the differentiation of human neural progenitor cells was shown by immunocytochemistry. RESULTS: Human neural progenitor cells developed into functional cells in the injured spinal cord and improved the recovery of injured spinal cord in both locomotor scores and electrophysiological parameters in rats. CONCLUSIONS: Human neural progenitor cells can treat injured spinal cord, which may provide a new cell source for research of clinical application.

Monoclonal side population progenitors isolated from human fetal pancreas.

The side population (SP) phenotype might represent a common molecular feature for a wide variety of stem cells. The aim of this study was to investigate whether monoclonal SP progenitor cells were established from human fetal pancreas. Islet-like cell clusters (ICCs) were isolated from human fetal pancreas. Monolayer epithelium-like cells were obtained from the ICCs and passaged thereafter. Single SP or non-SP cells were sorted from these cells at the sixth passage. The rate of clone formation was about 2.7% for the SP cells, whereas there was no clone formation for the non-SP cells. The SP cell clones were further expanded for more than 15 passages and induced for differentiation into cells with characteristics of pancreatic beta-cells. We show for the first time that the monoclonal SP progenitors are established from human fetal pancreas. Therefore, this study may offer a novel method to purify pancreatic progenitor cells from human tissues.

Nestin-positive progenitor cells isolated from human fetal pancreas have phenotypic markers identical to mesenchymal stem cells.

AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro. METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescence-activated cell sorting (FACS) and radioimmunoassay (RIA) were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 times in vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cell markers; (3) FACS analysis revealed that CD44, CD90 and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreatic-duodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intra-cellular insulin content was detected by RIA after the induction culture. CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulin-producing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.

[Directed differentiation of Balb/C mouse embryonic stem cells into pancreatic islet-like cell clusters in vitro: observation by atomic force microscope].

OBJECTIVE: To observe the morphological changes of Balb/C mouse embryonic stem cells following directed differentiation into pancreatic islet-like cell clusters (PICC) in vitro using atomic force microscope (AFM). METHODS: Balb/C mouse embryonic stem cells were first cultured into embryonic bodies (EBs) and allowed to differentiate spontaneously for 4 days. The cells were then transferred to gelatin-coated dishes for the EBs to attach and spread on the tissue culture plates, in the course of which a series of cell growth factors such as basic fibroblast growth factor (bFGF), insulin-like growth factor 1 (IGF-1) and nicotinamide were added into the culture medium at specific time points to induce directed differentiation of the stem cells into PICC. Immunocytochemistry was employed to detect the cells positive for insulin and glucagon, which were observed with AFM. RESULTS: The embryonic stem cells developed into cell clusters of different sizes, in which the cells were tightly arranged. Islet B cells were numerous in the center of clusters and darkly stained, but fewer in the peripherals with lighter stains. Islet A cells expressing glucagon were relatively fewer in the cell clusters, found mainly in the peripherals. Scanning of the insulin-positive clusters by AFM revealed large quantity of tissue fibers resembling nerve fibers that formed a reticular structure in disorderly arrangement. Numerous round granules were observed in the cytoplasm of almost identical sizes ranging from 0.5 to 1.0 mum in diameter. CONCLUSION: The cell clusters obtained by directed differentiation are mature in both morphology and function with also well organized structures.

Mutation analysis of PAX6 gene in a large Chinese family with aniridia.

BACKGROUND: Mutations in PAX6 gene have been shown to be the genetic cause of aniridia, which is a severe panocular eye disease characterised by iris hypoplasia. However, there is no study to do genetic analysis of aniridia, although there are several case reports in China. Here, we describe a mutation analysis of PAX6 in a large Chinese family with aniridia. METHODS: Genomic DNA from venous blood samples was prepared. Haplotype analysis was performed with two genetic markers (D11S904 and D11S935). Fourteen exons of the PAX6 gene were amplified from genomic DNA. Polymerase chain reaction (PCR) products of each exon were analysed by single strand conformational polymorphism (SSCP). The PCR products having an abnormal pattern were sequenced to confirm the mutation. RESULTS: Significant evidence for allele sharing in affected patients was detected suggesting that PAX6 mutation links to aniridia in this family. An extra band corresponding to exon 9 in PAX6 was found by single strand conformational polymorphism analysis in all the aniridia patients in this family, but not detected in the unaffected members. A mutation of C to T was detected by sequencing at the nucleotide 1080 that converts the Arg codon (CGA) to the termination codon (TGA). CONCLUSIONS: Aniridia is caused by a nonsense mutation of PAX6 gene in the large Chinese kindred. Genetic test is important to prevent the transmission of aniridia to their offsprings in the kindred by prenatal diagnosis.

[PAX6 mutation caused brain abnormalities in humans].

OBJECTIVE: To investigate the association between PAX6 mutation and brain abnormalities. METHODS: The brain structures of 18 affected patients and 6 normal controls in a large pedigree with a PAX6 mutation (c1080C-->T)were scanned with MRI assessing. RESULTS: Most of the affected patients showed brain abnormalities such as corpus callosum degeneration, broad cerebral ventricle grooves and broad olfactory grooves. CONCLUSION: Genetic defect of PAX6 gene may result in brain abnormalities.

[Expression of nestin and neurogenin 3 in the human fetal pancreas].

OBJECTIVE: To examine the expression of nestin and neurogenin 3 (Ngn3), the markers of pancreatic stem cells, in the human fetal pancreas. METHODS: The human fetal pancreas tissue of 12 and 14 weeks were examined for the expression of nestin and Ngn3 using the techniques of immunofluorescence dye and RT-PCR. RESULTS: Both nestin and Ngn3 expressed widely in 12 and 14 weeks before in human fetal pancreatic tissue. In these positive cells there was no co-expressing insulin or glucagon. There were nestin and Ngn3 co-expressing cells in ducts but not in the islets. The results of RT-PCR also indicated the expression of nestin and Ngn3. CONCLUSIONS: There was no expression of the markers of mature endocrine cells in the nestin and Ngn3 positive cells, and they were the marks of no-differentiation cells in the human fetal pancreatic tissue.

[Flow cytometry analysis and differentiation study of selected nestin positive cells].

OBJECTIVE: To verify the hypothesis that selected nestin positive cells derived from human fetal pancreas (according as medical ethnics) have surface markers similar to bone marrow mesenchymal stem cells (MSCs), and that these cells have multilineage potential. METHOD: The cell surface markers were determined by flow cytometry, and then the potential that these cells might be differentiated into adipocytes and osteoplasts were explored. RESULT: These cells have similar surface markers as MSCs of bone marrow origin. These cells was induced to differentiate into adipocytes and osteoplasts. CONCLUSION: Selected nestin positive cells derived from human fetal pancreas have certain characteristics of MSCs.

[Association of cytochrome P450 gene MSP1 polymorphism and risk of preterm].

OBJECTIVE: To investigate the association of cytochrome p450 gene (CYP1A1)MSP1 polymorphisms with preterm delivery. METHODS: Between July 1999 and June 2001, we conducted a case-control study using infant-parent triads including 247 families with full-term infants and 249 families with preterm delivery infants in Anqing, China. We extracted DNA from umbilical cord blood of the infants and vein blood of their parents,and performed PCR followed by restriction enzyme MspI digestion for genotyping the CYP1A1 gene MSP1 polymorphism. We used log-linear modeling to analyze the association of CYP1A1 gene polymorphism with the risk of preterm delivery. RESULTS: CYP1A1 gene C/C6235 increased the risk of preterm delivery both in infants (RR=1.80, 95% CI=1.02-3.18) and in their mothers (RR=1.82, 95% CI=1.11-2.98) significantly. There was no interaction between mothers' and children's CYP1A1 MSP1 genotypes. The variant alleles of CYP1A1 MSP1 of control triads accorded with Mendelian transmissions. CONCLUSION: Both infant and maternal CYP1A1 C/C6235 genotype both can increase the risk of preterm delivery in our study population, which suggests a possible role of human cytochrome P450 variability in the etiology of preterm delivery.

[Isolation, culture and multipotent differentiation of mesenchymal stem cells from human fetal livers].

OBJECTIVE: To isolate and culture mesenchymal stem cells (MSCs) from human fetal livers and describe their biological characteristics. METHODS: MSCs were acquired using an optimized method. Cell cycles and the immunophenotype of the cells were analyzed by flow cytometry. The osteogenic and adipogenic differentiations were induced and identified by specific stainings, and hepatic differentiation by morphology and RT-PCR. RESULTS: The target cells derived from human fetal livers adhered to the plate with fibroblast-like morphology, whose surface markers were CD90, CD44, CD147 positive, and CD34, CD45, HLA-DR negtive. In the differentiation study, these cells could be induced to differentiate into osteogenic, adipogenic and hepatocyte-like cells. CONCLUSION: Multipotent MSCs can be isolated and cultured from human fetal livers.

High-ratio differentiation of embryonic stem cells into hepatocytes in vitro.

BACKGROUND/PURPOSE: To date, in differentiating system of embryonic stem (ES) cells into hepatocytes, hepatic differentiation ratio was still not shown. Here, after investigating hepatic differentiation from ES cells, we determined the differentiation ratios of hepatocytes and studied how to improve the ratios in ES cell differentiating system. METHODS: Embryonic bodies (EBs) formed from ES cells for 5 days were plated onto culture dishes and some growth factors were added into medium for hepatic differentiation. Expressions of hepatic genes and proteins were analysed using reverse transcriptase-polymerase chain reaction, immunocytochemistry (ICC) and radioimmunoassay. The relative counts of hepatocyte-like cells among all EBs cells were analysed by flow cytometry by which hepatic differentiation ratios were determined. Then, we observed the spatial distribution of ICC-positive cells in EB cells cluster and isolated the cells of positive areas in other EBs clusters without ICC examined. At last, isolated cells were re-cultured with previous condition and hepatic differentiation ratios were also determined. RESULTS: The hepatic genes and proteins were, respectively, expressed in cytoplasm. Hepatic differentiation ratio was first determined at day 11 to be 12.1% and the level reached maximum to be 33.4% at day 21. In isolated cells culture system, hepatic genes and proteins expressed stronger than that expressed in EBs cluster and hepatic differentiation ratio was got to 72.6% at day 21. CONCLUSIONS: Isolating hepatocyte-like cells from EBs cell cluster and re-culturing them could produce hepatocytes with high differentiation ratio. This culture system may produce a new source of cell types for hepatocytes replacement therapies in hepatic failure.

[Transferrin receptor expression of human mesenchymal stem cells and in vitro tracking by autoradiography after transplantation in spinal cord].

OBJECTIVE: To determine transferrin receptor (TfR) expression of human mesenchymal stem cells (MSCs) in vitro and after transplantation in rabbit spinal cord,and to detect implanted MSCs by in vitro autoradiography. METHODS: Human mesenchymal stem cells (hMSCs) were isolated from fetal blood. Flow cytometry assay, immuno-fluorescent staining and receptor binding assay were used to determine TfR expression of hMSCs. Radioiodinated transferrin saturated with iron [(125)I-Tf(Fe)(2)] was used as tracer. The hMSCs transplanted in rabbit spinal cord was tracked by in vitro autoradiography. Diffusion of (125)I-Tf(Fe)(2) in spinal cord was examined with autoradiography. RESULTS: TfR expression of MSCs was demonstrated by flow cytometry assay, immuno-fluorescent staining and receptor binding assay in vitro. (125)I-Tf(Fe)(2) bound to hMSCs with a equilibrium dissociation constant (KD) of (0.98+/-0.12) nmol/L and a maximal density of binding sites (B(max)) of (107 702+/-6 226) sites per cell. Immuno-fluorescent staining showed that TfRs were expressed on hMSCs on the 2nd day but not be expressed on the 10th day post transplantation. Autoradiography showed distinct accumulation of (125)I-Tf(Fe)(2) but not (125)I-HSA at hMSCs implantation sites of spinal cord sections on the 2nd day post transplantation. (125)I-Tf(Fe)(2) had diffused into spinal cord 16 hours after incubation. CONCLUSION: Implanted hMSCs could be detected by in vitro autoradiography with (125)I-Tf(Fe)(2) on the 2nd day after being transplanted in spinal cord. To track implanted hMSCs with radionuclide imaging techniques in vivo, TfR was a suitable target for imaging and radioiodinated Tf(Fe)(2) was a feasible tracer.

[The ultrastructure study of membrane surface of the bone marrow CD34+ cells with the atomic force microscope].

Human CD34(+) hematopoietic cells, a distinctive cell population containing hematopoietic stem/progenitor cells (HSPC), have the capability to highly self-renewal, differentiation into all lineages of committed progenitor cells and reconstitution of both long-term hematopoiesis and immunefunctions after transplantation. CD34(+) hematopoietic cells from bone marrow (BM) recently have been employed for treating neoplastic and genetic disorders. This study was aimed to investigate membrane surface ultrastructures of bone marrow CD34(+) cell from mormal persons and leukemia patients and to compare their morphologic differences by using atomic force microscope (AFM). BM was collected from 5 normal donors and 6 leukaemia patients. All samples were layered on Ficoll-Paque gradients (specific gravity 1.077 g/ml) to separate the mononuclear cells. After that CD34(+) cells were purified by immuno-magnetic bead separation and evaluated with a FACS Calibur, these cells were detected by AFM of tapping mode inair. At lest 20 cells per samples were observed. The results showed that most of CD34(+) hematopoietic cells were like circle plate, the diameter was 10 - 14 microm. The surface of CD34(+) hematopoietic cell membrane was comparatively complex. The surface of CD34(+) hematopoietic cell membrane appeared as granular, with packed particles. With the region analysis function of IP2.1 software, the region of 2 microm x 2 microm was selected and four parameters of the surface (maximum peak-to-valley distance, average roughness, root-mean-squared roughness and mean height) were measured. Values of the 4 parameters showed that the characteristic parameters of CD34(+) HSPC from leukaemia were higher than that from normal person. It is concluded that AFM has specific advantages in analyzing cell membrane in the nanometer level and can gain more information. With the help of analysis software, AFM can be a helpful tool for fast leukaemic diagnosis and CD34(+) hematopoietic cells selection.