Number Of Pluripotent Stem Cells Research Articles

Detection of Cancer Stem Cells in Normal and Dysplastic/Leukemic Human Blood.

The hierarchical organization of the leukemic stem cells (LSCs) is identical to that of healthy counterpart cells. It may be split into roughly three stages: a small number of pluripotent stem cells at the top, fewlineage-restricted cells in the middle, and severalterminally differentiated blood cells at the bottom. Although LSCs can differentiate into the hematopoietic lineage, they can also accumulate as immature progenitor cells, also known as blast cells. Since blast cells are uncommon in healthy bloodstreams, their presence might be a sign of cancer. For instance, a 20% blast cutoff in peripheral blood or bone marrow is formally used to distinguish acute myeloid leukemia from myelodysplastic neoplasms, whichis essential to plan the patients' management. Many techniques may be useful for blast enumeration: one of them is flow cytometry, which can perform analyses on many cells by detecting the expression of cell surface markers. Leukemic and non-leukemic blast cells might indeed be characterized by the same surface markers, but these markers are usually differently expressed. Here we propose to use CD45, in combination with CD34 andother cell surface markers, to identify and immunophenotype blast cells in patient-derived samples.

A small-molecule approach towards the Fountain of Youth: chemically induced pluripotent stem cells.

Generation and regeneration as an answer to disease treatment has been around for some time. Yet never have we come so close to reaching such 'life-altering' capabilities. Today, the field of regenerative medicine research focuses on replacing non-functional or dead cells with healthy ones, in order to repair or regenerate tissues and organs to restore normal functions. Pluripotent stem cells have the ability of long-term self-renewal and possess the potential to differentiate to all kinds of functional cells in humans. Therefore, how to directly obtain a large number of pluripotent stem cells from patients in vitro, to be grown into differentiated specific tissues and organs, has become one of the most important topics. Six decades ago, Gurdon's group discovered that cell differentiation is a reversible process [1], laying down the foundation for cell reprogramming research. Commonly there are biological and chemical methods for the acquisition of pluripotent stem cells in vitro, which also aim to produce further differentiated specific tissues and organs. Fifteen years ago, Yamanaka's group first reported the acquisition of induced pluripotent stem cells (iPSCs) via overexpression of four transcription factors OSKM to the somatic cells [2]. Chemical reprogramming-using cell permeable small molecules to manipulate the cell fates-has also progressed significantly. Hongkui Deng at Peking University and his co-workers reported that a combination of small molecule compounds could induce pluripotent stem cells from mouse somatic cells with an induction efficiency as high as 0.2% in 2013 [3]. After long-term persistence and unremitting efforts, Deng's group announced the acquisition of chemically induced pluripotent stem cells (CiPSCs) from human fibroblasts through a step-wised chemical reprogramming strategy in 2022. This technology for preparing human CiPSCs solves the underlying technical bottleneck for the development of stem cells and regenerative medicine, and advances the application of cell reprogramming towards a new stage [4]. As the progress in human cell reprogramming led to sufficient resources of CiPSCs, chemically induced cell fate trans differentiation research also brought us surprises. Deng and colleagues not only demonstrated that small molecules can reprogram astrocytes into neurons in the adult mouse brain, which provides a potential approach for developing neuronal replacement therapies [5], but also constructed a bio-artificial liver device through directed differentiation of human pluripotent stem cells to hepatic cells [6]. Recently, Deng and colleagues established an efficient method for producing islet cells from human CiPSCs and demonstrated that these cells were able to ameliorate diabetes in non-human primates [7]. CiPSCs might be considered to have potential in the fields of cell therapy, drug screening and disease modeling, and are the most critical 'seed cells' in the field of regenerative medicine. Emerging as important regulators of cell fate, natural product small molecules and their derivatives have played an important role in Deng's work. NSR spoke to Hongkui Deng about the highlights and possibilities of the field.

Диференціювання індукованих плюрипотентних стовбурових клітин в кардіоміоцити в суспензійній культурі з постійним перемішуванням та в спеціальних планшетах з мікролунками

In order to enhance the differentiation of induced pluripotent cells into cardiomyocytes, we compared two methods of embryoid bodies formation: differentiation in rotating suspension culture and formation of embryoid bodies from a predetermined number of pluripotent stem cells in microwells of AggreWell plates. We used transgenic murine induced pluripotent stem cell line AT25. Cell line expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter (αMHC). We applied flow cytometry and fluorescence microscopyin order to test the efficiency of differentiation processes. Thus, differentiation of pluripotent stem cells in AggreWell plates without adding differentiation factors was more effective than differentiation in rotating suspension culture. However, we obtained the most amounts of cardiomyocytes on the 11-th day in rotating suspension culture with ascorbic acid, after we applied dorsomorfin, DMSO, ascorbic acid, G-CSF with the above-mentioned methods. The amount of GFP + cells was 2,71 ± 0,07%.

RNA-binding proteins and microRNAs in gastrointestinal epithelial homeostasis and diseases.

The epithelium of gastrointestinal (GI) mucosa is a rapidly self-renewing tissue in the body, and its homeostasis is preserved through strict regulation of cell proliferation and apoptosis. Epithelial cells originate from a small number of pluripotent stem cells, which divide to either renew themselves or become committed crypt cells. RNA-binding proteins (RBPs) and microRNAs (miRNAs) regulate gene expression at the posttranscriptional level and are recently shown to modulate GI mucosal growth and repair after injury. Here we highlight the roles of RBPs HuR, CUG-binding protein 1, AU-binding factor 1, and several GI epithelial-specific miRNAs in gut mucosal homeostasis and diseases and also further analyze the mechanisms through which RBPs and miRNAs modulate the stability and translation of target mRNAs.

A modified method for implantation of pluripotent stem cells under the rodent kidney capsule.

Teratoma formation, the standard in vivo pluripotency assay, is also frequently used as a tumorigenicity assay. A common concern in therapeutic stem cell applications is the tumorigenicity potential of a small number of cell impurities in the final product. Estimation of this small number is hampered by the inaccurate methodology of the tumorigenicity assay. Hence, a protocol for tumorigenicity assay that can deliver a defined number of cells, without error introduced by leakage or migration of cells is needed. In this study, we tested our modified transplantation method that allows for transplant of small numbers of pluripotent stem cells (PSCs) under the kidney capsule with minimal cell leakage. A glass capillary with a finely shaped tip and an attached mouth pipette was used to inject PSCs into the rodent kidney capsule. H9 embryonic and induced PSCs were tagged with Fluc and green fluorescence protein reporter genes and divided in different cell doses for transplantation. Bioluminescence imaging (BLI) on the day of surgery showed that the cell signal was confined to the kidney and signal intensity correlated with increasing transplant cell numbers. The overall cell leakage rate was 17% and the rodent survival rate was 96%. Teratoma formation was observed in rodents transplanted with cell numbers between 1 × 10(5)-2 × 10(6). We conclude that this modified procedure for transplanting PSCs under the kidney capsule allows for transplantation of a defined number of PSCs with significant reduction of error associated with cell leakage from the transplant site.

Human iPSC-derived miniature organs: a tool for drug studies.

In vitro organogenesis is now becoming a realistic goal of stem cell biology, as one can obtain an unlimited number of pluripotent stem cells through reprogramming technology. One practical challenge is to develop a four-dimensional (4-D) stem cell culture system whereby multiple progenitors communicate in a spatiotemporal manner, as observed during in vivo organogenesis. This perspective summarizes the potential for emerging culture platforms in the future application of induced pluripotent stem cell (iPSC)-derived miniature organs by recapitulating early embryogenesis.

A new paradigm for African American breast cancer involving stem cell differentiation.

Abstract Abstract #5051 Background: African American (AA) women have a higher incidence of breast cancer (BC) than white women before age 40, and are more likely to die from BC at every age. These differences are often attributed to socio-economic factors, however, there may be intrinsic biological differences in the breast tissue of AA and white women. For example, triple negative breast tumors are more prevalent in AA women than in non-Hispanic white women, although neither population is monolithic in its breast cancer phenotypes. We hypothesis that intrinsic biological differences between women of AA and European-white ancestry might be revealed by differences in growth and differentiation in vitro, reflecting underlying differences in the proportions and/or potency of breast epithelial stem cells.
 Methods: Our laboratory has developed a novel tissue engineering system for Human Mammary Epithelial Cells (HMEC), both normal and malignant. This system allows for long-term establishment of non-diseased primary cultures that begin as 3-dimensional attached “epispheres”, that are structures made up of 40-100 epithelial cells. These non-diseased epispheres subsequently differentiate into functional, organotypic branching ducts and lobules that demonstrate ESA and CK -18,-19 staining, lumen, polarized nuclei, desmosomes, microvilli on apical surfaces and casein secretion.
 Results: We have established primary HMEC cultures from 36/36 breast reduction mammoplasty tissues, including 9 AA donors matched in socioeconomic status. We have found: 1) The more children a woman had, the less likely her breast tissue was to form ductal structures in vitro. This finding is consistent with the idea that lactational differentiation decreases the number of pluripotent stem cells in the breast. 2) Pre-menopausal breast tissue was more likely to form ductal structures than post-menopausal tissue. 3) Race was a modifying factor in the ability to form ductal architecture in culture, significantly affecting the rate of differentiation. Putative mammary stem cells were analyzed by flow cytometry (CD44+, CD24-, MUC1-, α6 integrin+) and cloning. Stem cell proportions varied widely (range 0.5-15%), with no obvious association with race.
 Discussion: One interpretation of the hormonal risk factors for BC involves the susceptibility of undifferentiated breast tissue to transformation. In other systems, especially the gonads, interference with terminal differentiation is also associated with susceptibility to transformation. If AA breast tissue has more robust differentiation potential than that of white women, it may also contain stem cells more likely to transform into an aggressive tumor type. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5051.

MUC1* Mediates the Growth of Human Pluripotent Stem Cells

The MUC1 protein is aberrantly expressed on an estimated 75% of all human solid tumor cancers. We recently reported that a transmembrane cleavage product, MUC1*, is the predominant form of the protein on cancer cells [1]. Further, our evidence indicated that MUC1* functions as a growth factor receptor on tumor cells, while the full-length protein appeared to have no growth promoting activity. Here, we report that MUC1* acts as a growth factor receptor on undifferentiated human embryonic stem cells (hESCs). Cleavage of the full-length ectodomain to form MUC1*, a membrane receptor, appears to make binding to its ligand, NM23, possible. Unexpectedly, we found that newly differentiated cells no longer express the cleaved form, MUC1*, or its ligand, NM23. Newly differentiated stem cells exclusively present full-length MUC1. Antibody-induced dimerization of the MUC1* receptor on hESCs stimulated cell growth to a far greater degree than currently used methods that require the addition of exogenous basic fibroblast growth factor (bFGF) as well as factors secreted by fibroblast “feeder cells”. Further, MUC1* mediated growth was shown to be independent of growth stimulated by bFGF or the milieu of factors secreted by feeder cells. Stimulating the MUC1* receptor with either the cognate antibody or its ligand NM23 enabled hESC growth in a feeder cell-free system and produced pluripotent colonies that resisted spontaneous differentiation. These findings suggest that this primal growth mechanism could be utilized to propagate large numbers of pluripotent stem cells for therapeutic interventions.

Differences in Umbilical Cord Blood Units Collected during Cesarean Section, before or after the Delivery of the Placenta

Umbilical cord blood is largely employed as an alternative source of stem cells in the treatment of hemato-oncological diseases. Current results show that the success rate of purified umbilical cord blood engraftment is comparable to that obtained using bone marrow, and it is directly related to the number of pluripotent stem cells transplanted. The technique of fetal blood collection varies among different umbilical cord blood banks. Many authors collect umbilical cord blood during vaginal delivery, after placental detachment, while others collect it while the placenta is still within the uterus. In a previous randomized trial, we showed a greater collection of umbilical cord blood before placental detachment during vaginal delivery. The present study was performed to determine whether umbilical cord blood collection before placental detachment (group A) during cesarean section is superior to that after placental delivery (group B) puncturing the umbilical vein once and using a closed bag system. To accomplish this, 47 pregnant women subjected to cesarean section were enrolled in the study. Twenty-one of them were allocated to group A, while the remaining 26 formed group B. The volume of umbilical cord blood collected from the patients of group A was greater than that collected from patients of group B. The cord blood volume collected was 90.7 ± 6.0 versus 60.9 ± 13.7 ml; the cord blood nucleated cell number was 10.1 ± 1.2 × 10⁸ vs. 7.1 ± 0.8 × 10⁸; and the mean cord blood CD34+ cell number was 20.0 ± 6.0 × 10⁵ vs. 16.4 ± 2.4 × 10⁵, respectively.

Significance of the SCID Mouse Models for the Study of Normal and Leukemic Human Hemopoiesis and Gene Therapy

The hemopoietic system is organized as a hierarchy with all myeloid and lymphoid cells arising from a small number of pluripotent stem cells. As current in vitro methods do not assay stem cells, transplantation-based models for normal and leukemic human hemopoietic cells have been developed using immune-deficient mice as recipients. In vivo models were established for adult, neonatal, and fetal human hemopoiesis, for human leukemias and for genetic disorders and gene therapy. This transplantation system can be used to characterize and purify candidate human stem cell populations. Moreover, recent studies have provided strong support for the concept that there is a hierarchy of stem and progenitor cells for human leukemias. The SCID mouse models enable analysis of the biology of and metabolization of cyto-static drugs by leukemic stem cells. In addition, they are an important tool in developing and improving gene therapy protocols by assessing the efficiency of gene transfer into primitive genetically deficient stem cells following engraftment in immune-deficient mice. Potentials and limitations of the different approaches are discussed.

Age-Associated Changes in Antibody-Forming Cells (B Cells)

ConclusionDespite considerable experimental effort, it is not possible to state unequivocally that with aging there occur some intrinsic changes in B cells. We have some indication that the number of pre-B cells may be affected in the bone marrow of the aged. If these changes extend to a decrease of the actual number of pluripotent stem cells, then this would indeed contribute to a lack of self-renewing capacity and affect the total peripheral B cell population. In those instances when actual precursor cell frequencies have been enumerated, even though for the majority of antigen systems studied B cell precursor frequency decreases in the aged, the responding B cells showed no individual functional decline (i.e., amount of antibody synthesized). Evidence has been obtained that changes in regulatory mechanisms are responsible, in part, for the decline in the immune response of the aged. This is particularly obvious when VH gene usage in antibody is compared between the total available repertoire (i.e., fol...

Estimating the size of the dividing stem cell pool after allogeneic bone marrow transplantation

Currently the problem of estimation of the number of pluripotent stem cells reconstituting marrow grafts following bone marrow transplantation, or studies looking at questions of clonal dominance in hematopoietic cell populations, rely on indirect measurement and a simple application of the formula for the sampling variation of a binomial proportion. This approach, from a statistical viewpoint, can be seen to be flawed. It is very easily remedied though and only requires appropriate use of variance stabilizing transformations. These lead to a very simple estimator for the number of hematopoietic stem cells involved in repopulating the marrow and require little in the way of additional calculation. We give the distribution theory for this estimator as well as simple approximations for practical application. As an illustration we rework data recently gathered to address the question as to whether or not reconstitution of marrow grafts in the clinical setting is oligoclonal.

Effects of prolonged nitrous oxide exposure on hemopoietic stem cells in mice.

The effects of prolonged exposure to nitrous oxide on the hemopoietic progenitor cells in bone marrow and spleen in mice were investigated. Fifty percent nitrous oxide caused a marked decrease in the number of pluripotent stem cells (CFU-S) and granulocyte-macro-phage progenitor cells (GM-CFC) in the spleen, whereas it caused only a slight decrease in these cells in the bone marrow. These results suggest that prolonged exposure to nitrous oxide induces damage in the splenic hemopoiesis in mice.

Murine recovery from busulfan‐induced hematopoietic toxicity as assessed by three assays for colony‐forming cells

AbstractThese studies were designed to explore further the interrelationships between various stem cell and other hematopoietic compartments of the mouse. Busulfan (BU) has been shown by others to have a profound effect on the transplantable pluripotent stem cells capable of forming colonies on spleens (CFU) of lethally irradiated recipients – more so than on cells of differentiated compartments. Another presumed assay of pluripotent stem cells consists of counting colonies on spleens of nontransplanted sublethally irradiated mice as a quantitation of endogenous colony‐forming units (E‐CFU). We examined and compared BU‐induced damage and recovery as measured by E‐CFU, CFU, CFU‐C, total cellularity of marrow, peroxidase‐positive cells per marrow, spleen weight, radioactive iron uptake into spleen and marrow, and hematocrit. E‐CFU were severely damaged by BU, but recovery was significantly more rapid than that measured for CFU. These results were compatible with other data suggesting that cycling stem cells are more likely to survive radiation and less likely to survive removal and transplantation. Thus, the actual number of pluripotent stem cells lies between the CFU and E‐CFU values. Damage to more mature granulocytic and erythrocytic compartments lagged behind damage to E‐CFU and CFU. Abortive rises in these more mature compartments were observed, which suggested limited but distinct self‐replication activity of “stem cells” more differentiated than the CFU, E‐CFU systems. Time of final recovery of the damaged hematopoietic system was compatible with a concept of recovery of pluripotent stem cell system(s) preceding recovery of more mature compartments.

Thiamphenicol as an inhibitor of early red cell differentiation.

The effects of an in vivo treatment of mice with thiamphenicol on stem cells are shown. Thiamphenicol causes a drastic depletion of erythroid precursors in the marrow. Mitochondrial protein synthesis is inhibited resulting in a severely reduced cell proliferation. The number of pluripotent stem cells in the marrow did not decrease. In the spleens of thiamphenicol-treated mice a strong reduction of stem cells was found. The sedimentation behaviour of stem cells from anemic and thiamphenicol-treated mice was similar. The pluripotent stem cells from thiamphenicol-treated mice were in a low cycling state, despite a very high erythropoietin level. Under these circumstances a partial commitment of the pluripotent stem cells into the erythroid direction was observed.

Some effects of chemotherapeutic drugs on bone marrow stem cells

The non-Hodgkin lymphoma chemotherapy protocol used at the Gustave-Roussy Institute was adapted, in terms of drug doses and interval between doses, to normal CBA mice. The numbers of pluripotential stem cells (CFU-S), unipotential stem cells (CFC), differentiated bone marrow cells, and circulating white cells were determined. Eight hours after each drug of the first chemotherapy cycle the number of pluripotent stem cells decreased while the proportion of these cells in DNA synthesis increased. Six hours after the end of each complete cycle, the stem cell compartments were found to be considerably depleted, and they were not completely restored when the next cycle was begun, while the other hematologic compartments were completely restored at this time.

Hemopoiesis in diffusion chambers in strontium-89 marrow-ablated mice.

SummaryThe numbers of pluripotent stem cells (CFU-S) and of the more differentiated granulocyte/macrophage elements in diffusion chambers (DCs) implanted into the peritoneal cavities of radio-89Sr-marrow-ablated mice are increased as compared to those in DCs implanted into cold-88 Sr-mar-row-ablated mice. These findings suggest that there is a systemic humoral response capable of stimulating hemopoiesis even in mice with aplastic marrows and whose hemopoiesis is localized to their spleens. The magnitude of this response and the promptness with which the response is manifest in DC growth suggests that marrow aplasia induced by 89Sr provides a stronger stimulus for proliferation of cells in DCs than does either cyclophosphamide or lethal external whole-body irradiation.

Effects of allogeneic stimulations on the proliferation and differentiation of the hemopoietic stem cell.

The effects of allogeneic stimulatins on the hemopoietic system were investigated. Spleen cells of mice activated in vivo with alloantigens were able to produce highly active colony-stimulating factor in the culture medium without any stimulation. But spleen cells of these mice could not release colony-stimulating factor after treatment in vitro with anti-theta serum and complement. The number of granulopoietic progenitor cells (colony-forming unit-culture) in both bone marrow and spleen cells of mice treated with such procedures was significantly greater than that of the control, and the number of pluripotent stem cells (colony-forming unit-spleen) of these mice was markedly increased compared with that of the control, especially in spleen cells. These experiments suggest that T lymphocytes activated in vivo with alloantigens may release active substances in the differentiation and proliferation of hemopoietic stem cells.

Erythropoietic progenitors capable of colony formation in culture: state of differentiation.

AbstractThe differentiated state of mouse erythropoietic progenitor cells (CFU‐E), detected by their ability to form erythropoietin‐dependent colonies in vitro, has been investigated. Transfusion‐induced plethora was found to reduce the population size of CFU‐E in both spleen and femoral marrow, which indicates that a significant number of CFU‐E arise by differentiation processes that are themselves erythropoietin‐dependent. Individual spleen colonies were found to be heterogeneous in their content of CFU‐E, and the numbers of CFU‐E per colony were not correlated either positively or negatively with the numbers of granulocyte‐macrophage progenitors (CFU‐C) present in the same colonies. The absence of a negative correlation between CFU‐E and CFU‐C indicates that the erythropoietic and granulopoietic pathways of differentiation are not mutually exclusive within individual spleen colonies. The numbers of CFU‐E per spleen colony were also found to vary independently of the numbers of pluripotent stem cells (CFU‐S) per colony; in contrast, as found previously, the numbers of CFU‐C and CFU‐S per colony were positively correlated. These results indicate that more randomizing events separate CFU‐E from CFU‐S than separate CFU‐C from CFU‐S, and are consistent with the view that CFU‐E occupy a position on the erythropoietic pathway of differentiation that is more remote from the pluripotent stem cells than is the corresponding position of CFU‐C on the granulopoietic pathway.