Cobblestone area-forming cells in human cord blood are heterogeneous and differ from long-term culture-initiating cells

The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However, comparing the two assays, cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells, p = 0.0001). Overall, 60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as "false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells, as expected, gave very similar readouts in the two assays, with 4,084 and 3,468/10(5) cells being CAFC and LTC-IC, respectively. CD34(-)/lineage(-) cells did not form cobblestones or become CD34(+) on stroma or in cytokine culture. Human CB MNC contain a population of mature lineage(+) cells, possibly mature T or B cells, which, although producing cobblestone areas (CA), are not functional LTC-IC. The CAFC readout by this method, therefore, is unreliable for estimation of primitive hematopoietic cells by limiting dilution analysis in whole human CB or MNC and also may not detect CD34(-) CA stem cells.     

粒细胞集落刺激因子动员对供者外周血细胞树突状细胞亚群的影响

目的：研究粒细胞集落刺激因子(G-CSF)动员对外周血单个核细胞（PBMNC）中树突状细胞（DC）亚群及其功能的影响。方法：以CD34^-Lin^- HLA-DR⁺细胞群设门4色荧光分析方法检测25例无关供者G-CSF动员前、后外周血细胞DC亚群；ELISA检测其血清相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4活性，并分析DC₁/DC₂（CD11c⁺CD123^- /CD11c^-CD123⁺）比值与CD34⁺细胞含量的相关性。结果：G-CSF动员后, 供者PBMNC 的DC₁/DC₂比值显著低于动员前（P<0.05）；DC₁/DC₂与CD34⁺细胞含量呈负相关（r=-0.438, P<0.05），CD34⁺细胞/MNC≥0.4%时， DC₁/DC₂倒置；而血清相关细胞因子IL-12p40、IL-10、IFN-γ、IL-4水平无显著改变（P>0.05）； DC₂ HLA-DR表达上调而CD83不表达。结论： G-CSF动员后，供者外周血CD34⁺细胞数量增加的同时，DC₂数量也增加，这些DC₂尽管HLA-DR表达上调，但仍处前体细胞状态，不直接分泌或调节Th2细胞分泌免疫抑制因子。     

Allogeneic stem cell transplantation with peripheral blood stem cells mobilized by pegylated G-CSF.

Mobilization of stem cells with pegylated granulocyte colony-stimulating factor (peg-G-CSF) modulates donor T- and natural killer T-cell (NKT-cell) functions, thus separating graft-versus-host from graft-versus-leukemia disease in animal models. We report a phase I/II study that analyzed the feasibility of mobilizing stem cells from normal donors with peg-G-CSF and the ability of these cells to restore hematopoiesis in allogeneic transplant recipients after myeloablative conditioning. Administration of 6 mg of peg-G-CSF resulted in suboptimal stem cell mobilization, with a peak peripheral blood CD34+ count of 29+/-5/microL. Apheresis 4 days after peg-G-CSF yielded 2.7+/-.4x10(6) CD34+ cells/kg recipient weight, and all donors required a second collection on day 5 to yield a total of 4.2+/-.5x10(6) CD34+ cells/kg recipient weight. After escalation of the dose to 12 mg, the peak CD34+ count was 99+/-11/microL and 12 of 13 donors collected sufficient stem cells for transplantation in a single apheresis (8.9+/-1.4x10(6) CD34+ cells/kg recipient weight). Late transient increases in serum hepatic transaminases were noted, but other side effects (predominantly bone pain) were otherwise similar to those seen in donors mobilized with standard G-CSF. Median neutrophil and platelet engraftments occurred on days 18 and 14, respectively, after transplantation and were identical to those seen with in recipients of grafts mobilized with standard G-CSF. With a median follow-up of 357 days, the incidence of grade II-IV acute graft-versus-host disease was 50% and there have been no relapses to date. Mobilization of stem cells with peg-G-CSF in normal donors is feasible and 12 mg results in mobilization characteristics similar to those of standard G-CSF.     

Proximal tubular cells contain a phenotypically distinct,scattered cell population involved in tubular regeneration

Regeneration of injured tubular cells occurs after acute tubular necrosis primarily from intrinsic renal cells. This may occur from a pre‐existing intratubular stem/progenitor cell population or from any surviving proximal tubular cell. In this study, we characterize a CD24‐, CD133‐, and vimentin‐positive subpopulation of cells scattered throughout the proximal tubule in normal human kidney. Compared to adjacent ‘normal’ proximal tubular cells, these CD24‐positive cells contained less cytoplasm, fewer mitochondria, and no brush border. In addition, 49 marker proteins are described that are expressed within the proximal tubules in a similar scattered pattern. For eight of these markers, we confirmed co‐localization with CD24. In human biopsies of patients with acute tubular necrosis (ATN), the number of CD24‐positive tubular cells was increased. In both normal human kidneys and the ATN biopsies, around 85% of proliferating cells were CD24‐positive – indicating that this cell population participates in tubular regeneration. In healthy rat kidneys, the novel cell subpopulation was absent. However, upon unilateral ureteral obstruction (UUO), the novel cell population was detected in significant amounts in the injured kidney. In summary, in human renal biopsies, the CD24‐positive cells represent tubular cells with a deviant phenotype, characterized by a distinct morphology and marker expression. After acute tubular injury, these cells become more numerous. In healthy rat kidneys, these cells are not detectable, whereas after UUO, they appeared de novo – arguing against the notion that these cells represent a pre‐existing progenitor cell population. Our data indicate rather that these cells represent transiently dedifferentiated tubular cells involved in regeneration. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Homing-associated cell adhesion molecules and cell cycle status on the nucleated cells in the bone marrow, mobilized peripheral blood and cord blood

Homing-associated cell adhesion molecules (H-CAM) on the CD34+ cells play an important role for the engraftment process following hematopoietic stem cell transplantation (HSCT). However, it seems that not only CD34+ cells but also other nucleated cells (NCs) with H-CAM could be implicated in the engraftment process and the proliferation of hematopoietic stem cells. We investigated the differences of HCAM and cell cycle status on the NCs in cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PB). The proportions of CXCR4+ cells within the NC populations were greater in CB than in PB or BM (p=0.0493), although the proportions of CXCR4+, CD44+, and CD49d+ cells within the CB CD34+ cell populations were same within BM or PB. A lower proportion of CD34+CD49d+ cells within the CD34+ cell populations was more noted in CB than in PB or BM (p=0.0085). There were no differences in cell cycle status between CB and BM or PB. Our results suggest that the migrating potential of CB would be enhanced with increased CXCR4 expression on the NCs, but the adhesion potential of CB CD34+ cells would be less than that of PB and BM. These findings may help explain why the lower cell dose is required and engraftment is delayed in cord blood stem cell transplantation.     

Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood.

Cord blood (CB) and autologous mobilized peripheral blood stem/progenitor cells (PBSC) are now used widely for clinical transplantation. We characterized the short-term (<8 weeks) and long-term (>8 weeks) engraftment in NOD/SCID mice resulting from transplanted CD34+ cells from these two sources. We also quantified the frequency of long-term engrafting cells, and the average proliferative capacity of individual engrafting cells by a competitive repopulation assay with binomial variance-covariance modeling. We found that 0.5 million CD34+ CB cells were able to generate sustained, high-level, multilineage human hematopoiesis, whereas a sixfold higher number of CD34+ PBSC (3 million) from cancer patients undergoing chemotherapy generated comparable short-term, but much lower sustained multilineage human hematopoiesis after transplantation. In comparison to CD34+ cells from PBSC from cancer patients, long-term engrafting cells were approximately eightfold enriched in CB CD34+ cells, and each CB long-term engrafting cell had an approximately 15-fold higher multilineage proliferative capacity. Thus, the number and function of transplantable hematopoietic cells were remarkably different between these two sources of stem/progenitor cells.     

K562细胞株侧群细胞中部分白细胞分化抗原的表达

背景：白血病侧群细胞表型的研究对于理解肿瘤细胞的异质性和起源、分子标记和靶向治疗等都有积极意义。 目的：鉴定人慢性粒细胞白血病细胞株K562中是否存在侧群细胞,并观察侧群细胞亚群与非侧群细胞亚群中部分白细胞分化抗原的表达差异。 方法：采用流式细胞术检测K562细胞株中是否存在侧群细胞;并进一步分析K562侧群细胞和非侧群细胞两亚群间CD34+、CD34+CD38-、CD34+CD38+、HLA-DR+细胞的表达情况。 结果与结论：经Hoechst33342染色,流式细胞仪分析结果显示在K562中存在侧群细胞,这部分细胞比例少,为(2.7±0.5)%;统计学分析侧群细胞和非侧群细胞亚群中CD34⁺、CD34⁺CD38^-细胞表达率差异有显著性意义,而CD34⁺CD38⁺细胞表达率和HLA-DR+细胞表达率差异均无显著性意义;侧群细胞和非侧群细胞相比在分化抗原表达上有异质性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Multipotential mesenchymal stem cells are mobilized into peripheral blood by hypoxia

MSCs constitute a population of multipotential cells giving rise to adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells. It remains unclear whether MSCs can be isolated from adult peripheral blood under stationary conditions and whether they can be mobilized in a way similar to hematopoietic stem cells. In this report, we show that MSCs are regularly observed in the circulating blood of rats and that the circulating MSC pool is consistently and dramatically increased (by almost 15-fold) when animals are exposed to chronic hypoxia. The immunophenotype and the adipocytic, osteoblastic, and chondrocytic differentiation potential of circulating MSCs were similar to those of bone marrow MSCs. Hypoxia-induced mobilization appears to be specific for MSCs since total circulating hematopoietic progenitor cells were not significantly increased. Our data provide an in vivo model amenable to analysis of MSC-mobilizing factors.     

Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood

Thrombocytopenia is a problematic and potentially fatal occurrence after transplantation of cord blood stem cells. This problem may be alleviated by infusion of megakaryocyte progenitor cells. Here, we compared the ability of hematopoietic progenitor cells obtained from cord blood and expanded in culture to that of mobilized peripheral blood cells. The CD34(+) cells were plated for 10 days in presence of thrombopoietin (TPO) alone and combined with stem cell factor (SCF), Flt3-ligand (FL), interleukin-3 (IL-3), IL-6, and IL-11. Cells were analyzed for the CD41 and CD42b expression and for their ploidy status. Ex vivo produced platelets were enumerated. We show that (1) TPO alone was able to induce differentiation of CD34(+) cells into CD41(+) cells, with limited total leucocyte expansion; (2) the addition of SCF to TPO decreased significantly CD41(+) cell percentage in CB, but not in MPB; and (3) in CB, the addition of FL, IL-6, and IL-11 to TPO increased the leukocyte expansion with differentiation and terminal maturation into MK lineage. In these conditions, high numbers of immature CD34(+)CD41(+) MK progenitor cells were produced. Our results thereby demonstrate a different sensitivity of CB and MPB cells to SCF, with limited CB MK differentiation. This different sensitivity to SCF (produced constitutively by BM stromal cells) could explain the longer delay of platelet recovery after CB transplant. Nevertheless, in CB, the combination of TPO with FL, IL-6, and IL-11 allows generation of a suitable number of immature MK progenitor cells expressing both CD34 and CD41 antigens, which are supposed to be responsible for the platelet recovery after transplantation.     

The late dividing population of gamma-retroviral vector transduced human mobilized peripheral blood progenitor cells contributes most to gene-marked cell engraftment in nonobese diabetic/severe combined immunodeficient mice

We used the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model to assess the repopulation potential of subpopulations of mobilized human CD34+ peripheral blood progenitor cells (PBPC). First, PBPC were transduced with gamma-retrovirus vector RD114-MFGS-CFP, which requires cell division for successful transduction, at 24 hours, 48 hours, and 72 hours to achieve 96% cyan fluorescent protein (CFP)-positive cells. Cells were sorted 12 hours after the last transduction into CFP-positive (divided cells) and CFP-negative populations. CFP-positive cells were transplanted postsort, whereas the CFP-negative cells were retransduced and injected at 120 hours. The CFP-negative sorted and retransduced cells contained markedly fewer vector copies and resulted in a 32-fold higher overall engraftment and in a 13-fold higher number of engrafted transgene positive cells. To assess cell proliferation as an underlying cause for the different engraftment levels, carboxyfluorescein succinimidyl ester-labeling of untransduced PBPC was performed to track the number of cell divisions. At 72 hours after initiation of culture, when 95% of all cells have divided, PBPC were sorted into nondivided and divided fractions and transplanted into NOD/SCID mice. Nondivided cells demonstrated 45-fold higher engraftment than divided cells. Late dividing PBPC in ex vivo culture retain high expression of the stem cell marker CD133, whereas rapidly proliferating cells lose CD133 in correlation to the number of cell divisions. Our studies demonstrate that late dividing progenitors transduced with gamma-retroviral vectors contribute most to NOD/SCID engraftment and transgene marking. Confining the gamma-retroviral transduction to CD133-positive cells on days 3 and 4 could greatly reduce the number of transplanted vector copies, limiting the risk of leukemia from insertional mutagenesis. Disclosure of potential conflicts of interest is found at the end of this article.     

Proliferation and telomere length in acutely mobilized blood mononuclear cells in HIV infected patients

The aim of the study was to investigate the mobilization of T cells in response to a stressful challenge (adrenalin stimulation), and to access T cells resided in the peripheral lymphoid organs in HIV infected patients. Seventeen patients and eight HIV seronegative controls received an adrenalin infusion for 1 h. Blood was sampled before, during and 1 h after adrenalin infusion. Proliferation and mean telomere restriction fragment length (telomeres) of blood mononuclear cells (BMNC) and purified CD8+ and CD4+ cells were investigated at all time points. In patients, the proliferation to pokeweed mitogens (PWM) was lower and decreased more during adrenalin infusion. After adrenalin infusion the proliferation to PWM was restored only in the controls. In all subjects telomeres in CD4+ cells declined during adrenalin infusion. Additionally, the patients had shortened telomeres in their CD8+ cells, and particularly HAART treated patients had shortened telomeres in all cell-subtypes. The finding that patients mobilized cells with an impaired proliferation to PWM during and after adrenalin infusion has possible clinical relevance for HIV infected patients during pathological stressful conditions, such as sepsis, surgery and burns. However, this study did not find a correlation between impaired proliferation and telomeres. It is concluded that physiological stress further aggravates the HIV-induced immune deficiency.     

Evidence that very small embryonic-like stem cells are mobilized into peripheral blood

Recently, we identified in murine adult tissues, including bone marrow, a population of very small embryonic-like (VSEL) stem cells. Here, we provide further evidence that under steady-state conditions these cells circulate at very low levels in peripheral blood (PB) ( approximately 100-200 cells/ml) and could be additionally mobilized during pharmacological granulocyte-colony-stimulating factor-induced or stress-related mobilization, as demonstrated in a model of toxic liver or skeletal muscle damage induced by injection of carbon tetrachloride or cardiotoxin, respectively. The number of circulating VSEL stem cells under steady-state conditions in PB of 2-month-old animals was five times higher than that in 1-year-old mice. In conclusion, this study supports a hypothesis that VSEL stem cells are a mobile pool of primitive stem cells that could be released from the stem cell niches into PB. Further studies are needed, however, to see whether the level of these cells circulating in PB could become a prognostic indicator to assess the regenerative potential of an adult organism and/or clinical outcome from an injury. Disclosure of potential conflicts of interest is found at the end of this article.     

Transcriptional profiling of mammary gland side population cells

Similar to the bone marrow, the mammary gland contains a distinct population of Hoechst-effluxing side population cells, mammary gland side population cells (MG-SPs). To better characterize MG-SPs, their microarray gene profiles were compared to the remaining cells, which retain Hoechst dye (mammary gland non-side population cells [MG-NSPs]). For analysis, Gene Ontology (GO) that describes genes in terms of biological processes and Ontology Traverser (OT) that performs enrichment analysis were used. OT showed that MG-SP-specific genes were enriched in the GO categories of cell cycle regulation and checkpoints, multidrug-resistant transporters, organogenesis, and vasculogenesis. The MG-NSP-upregulated genes were enriched in the GO category of cellular organization and biogenesis, which includes basal epithelial markers, p63, smooth muscle actin, myosin, alpha6 integrin, cytokeratin (CK) 14, and luminal markers CK8 and CD24. Additional studies showed that a higher percentage of MG-SPs exist in the G1 phase of the cell cycle compared with the MG-NSPs. G1 cell cycle block of MG-SPs may be explained by higher expression of cell cycle-negative regulatory genes such as transforming growth factor-beta2, insulin-like growth factor binding protein-5, P18(INK4C), and wingless-5a (Wnt-5a). Accordingly, a smaller percentage of MG-SPs expressed nuclear beta-catenin, possibly as a consequence of the higher expression of Wnt-5a. In conclusion, microarray gene profiling suggests that MG-SPs are a lineage-deficient mammary gland subpopulation expressing key genes involved in cell cycle regulation, development, and angiogenesis.     

Characterization and localization of side population cells in mouse skin

Recently, the detection of side population (SP) cells, which have the ability to strongly efflux Hoechst 33342 fluorescence dye, has attracted attention as a method of stem cell isolation. We identified SP cells from mouse skin using the same method as from bone marrow. This population almost completely disappeared after treatment with the calcium channel blocker verapamil. SP cells were mainly localized in the epidermis, with a few in the dermis. The ratio of SP cells decreased as the mouse became older. Surface marker analysis revealed that the sorted SP cells expressed alpha6-integrin, beta1-integrin, Sca-1, keratin 14, and keratin 19, which are proliferating and progenitor cell markers, at levels higher than in non-SP cells, while they expressed E-cadherin, CD34, and CD71 at lower levels. The expression of breast cancer resistance protein 1 (BCRP1), which participates in dye efflux, was expressed at high levels at both the protein and mRNA level in sorted SP cells. Immunohistochemical analysis showed that BCRP1 was expressed in the basal layers and hair bulge regions of mouse skin. BCRP1 mRNA was found in basal layers and hair follicles of newborn skin by in situ hybridization. These results indicate that the localization of BCRP1-positive cells is compatible with that of keratinocyte stem cells. Based on the close relationship between BCRP1 and the SP cell phenotype, we conclude that keratinocyte stem cells are closely related to the SP- or BCRP1-positive cells.     

26例动员外周血干细胞的程控降温冷冻保存效果评价

目的评价程控降温冷冻保存高浓度外周血干细胞的效果。方法采集动员的患者外周血干细胞,用程控降温系统降温至-80℃后保存于液氮中。冷冻前和解冻后均做有核细胞计数、粒单-集落形成单位(colonyformingunit-granulocyte/macrophage,CFU-GM)培养和苔盼蓝拒染率实验。结果冷冻的外周血有核细胞终浓度平均为1.405×108/ml,融化后有核细胞和CFU-GM回收率分别为80.64%±11.51%和94.01%±25.42%;拒染率为84.69%±8.72%。26例患者除1例死于预处理毒性不能进行评价外,余者均获得快速稳定造血功能重建。结论程控降温冷冻保存高浓度的外周血干细胞是一种安全有效的方法。     

Immunologic profiles of effector cells and peripheral blood stem cells mobilized with different hematopoietic growth factors

BACKGROUND: Peripheral blood stem cells (PBSC) have become the preferred source of stem cells for autologous transplantation because of the technical advantage and the shorter time to engraftment. Mobilization of CD34(+) cells into the peripheral blood can be achieved by the administration of G-CSF or GM-CSF, or both, alone or in combination with chemotherapy. G-CSF and GM-CSF differ somewhat in the number and composition of CD34(+) cells and effector cells mobilized to the peripheral blood. However, the molecular mechanism underlying the release and engraftment of CD34(+) cells is poorly understood. PURPOSE: The purpose of this review is to give a recent update on the type and immunological properties of effector cells and CD34(+) cells mobilized by the different growth factors with emphasis on A) mobilization of T cells, natural killer cells, and dendritic cells; B) coexpression of adhesion molecules such as VLA-4 and L-selectin in mobilized PBSC collection, and C) coexpression of CXCR4-the receptor for the stromal-derived differentiation factor 1-with latest information shedding light on the molecular mechanism underlying the release and subsequent engraftment of CD34(+) cells. CONCLUSIONS: A) The reported suppression of T cell and NK cell functions in PBSC apheresis collections in patients primed with G-CSF or GM-CSF is controversial and may merely reflect low effector cell activity before mobilization. B) A decrease in the expression of adhesion molecules such as VLA-4 and L-selectin is a necessary requirement for the release of CD34(+) cells to the peripheral blood. C) A decrease in the expression of CXCR4 is a necessary requirement for the release of CD34(+) cells to the peripheral blood and correlates with mobilization success.     

Characterization of side population cells from human airway epithelium

The airway epithelium is the first line of contact with the inhaled external environment and is continuously exposed to and injured by pollutants, allergens, and viruses. However, little is known about epithelial repair and in particular the identity and role of tissue resident stem/progenitor cells that may contribute to epithelial regeneration. The aims of the present study were to identify, isolate, and characterize side population (SP) cells in human tracheobronchial epithelium. Epithelial cells were obtained from seven nontransplantable healthy lungs and four asthmatic lungs by pronase digestion. SP cells were identified by verapamil-sensitive efflux of the DNA-binding dye Hoechst 33342. Using flow cytometry, CD45(-) SP, CD45(+) SP, and non-SP cells were isolated and sorted. CD45(-) SP cells made up 0.12% +/- 0.01% of the total epithelial cell population in normal airway but 4.1% +/- 0.06% of the epithelium in asthmatic airways. All CD45(-) SP cells showed positive staining for epithelial-specific markers cytokeratin-5, E-cadherin, ZO-1, and p63. CD45(-) SP cells exhibited stable telomere length and increased colony-forming and proliferative potential, undergoing population expansion for at least 16 consecutive passages. In contrast with non-SP cells, fewer than 100 CD45(-) SP cells were able to generate a multilayered and differentiated epithelium in air-liquid interface culture. SP cells are present in human tracheobronchial epithelium, exhibit both short- and long-term proliferative potential, and are capable of generation of differentiated epithelium in vitro. The number of SP cells is significantly greater in asthmatic airways, providing evidence of dysregulated resident SP cells in the asthmatic epithelium. Disclosure of potential conflicts of interest is found at the end of this article.     

Cells with hematopoietic activity in the mouse placenta reside in side population

The discovery of a major hematopoietic stem cell pool in midgestation mouse embryo has defined the placenta as an important hematopoietic anatomical site. In this study, we examined the flow cytometric pattern of mouse placenta cells on embryonic days (E) 10.5 to E15.5, in view of CD45 and c‐Kit expression. We also determined which population of these cells shows differentiation potential toward multiple hematopoietic lineages by performing coculture with OP9 stromal cells and colony‐forming assay in methylcellulose. Only CD45⁺c‐Kit⁺ population showed the ability to form hematopoietic colonies including multiple lineages. To distinguish which fraction of placenta cells have the hematopoietic activity, we used GFP transgenic mice in which the fetal part of the placenta is GFP positive and the maternal part is GFP negative. E11.5 and E13.5 CD45⁺c‐Kit⁺ placental cells that have ability to form hematopoietic colonies are the fetal GFP positive placental cells. E11.5 and E13.5 CD45⁺c‐Kit⁺ placental cells that have an ability to form hematopoietic colonies mainly reside in Hoechst dye‐effluxing side population area (SP). Taken together, in the placenta of mouse embryo, we conclude that SP cells in the CD45⁺c‐Kit⁺ fetal placental cells have the ability to form hematopoietic colonies.     

High frequency of fetal cells within a primitive stem cell population in maternal blood

BACKGROUND: During pregnancy, fetal cells enter the maternal bloodstreamresulting in fetal cell microchimerism. The fetal cells persistin the mother for decades and colonize a variety of maternalorgans. They are associated with maternal autoimmune diseasesand may also participate in tissue repair. The identity of themicrochimeric cells is not certain but they must be able topersist long-term and have potential for multitissue differentiation. METHODS AND RESULTS: Here we tested the hypothesis that the fetal microchimeric cellsare primitive stem cells, represented by CD34+ adherent cells,which have a wide potential for differentiation. We isolatedthese stem cells from the blood of pregnant females (n = 25)and detected fetal cells of the correct gender, using fluorescencein situ hybridization, in a high proportion (71% male fetusesand 90% female fetuses; false positive rate 11%, false negativerate 29%) of cases. By RT–PCR, we demonstrated that thecells express Oct-4, Nanog and Rex-1. No fetal cells were detectedin the mononuclear or total CD34+ cell populations but highfrequencies (mean 11.8%) of fetal cells were detected in theadherent CD34+ cell population. CONCLUSIONS: These results identify adherent CD34+ stem cells as candidatefetal microchimeric cells, which are capable of sustaining thefetal cell population in the long term and have the abilityto colonize multiple tissues and organs.     

A novel stem cell source for vasculogenesis in ischemia: subfraction of side population cells from dental pulp

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31(-);CD146(-) SP cells, demonstrating CD34+ and vascular endothelial growth factor-2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF-A, G-CSF, GM-CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell-based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.