The progenitor cell inhibition assay to measure the anti-leukemic reactivity of T cell clones against acute and chronic myeloid leukemia

Adoptive immunotherapy with donor T lymphocytes may be used as a treatment for relapsed leukemia after allogeneic hematopoietic stem cell transplantation (SCT). In vitro selected and expanded anti-leukemic T cells may be more effective in inducing a response in vivo. To identify the anti-leukemic reactivity of in vitro generated T cells, standard target cell read-out assays like the 51Cr-release assay are not always appropriate. We developed an assay in which the ability of T cells to antigen specifically inhibit the in vitro growth of leukemic progenitor cells in the presence of cytokines can be measured. This assay allows the evaluation of the cytolytic or suppressive potential of leukemia reactive T cells for prolonged periods of time. The assay is based on inhibition of [3H]thymidine incorporation by the leukemic progenitor cells induced by multiple hematopoietic growth factors. T cell clones with a known specificity were used to compare the analytic potential of the new assay with those of other cytotoxicity assays. Based on the results of the T cell clones, a modification of a limiting dilution assay was developed to identify anti-leukemic allogeneic T cells in HLA identical donor-recipient combinations selected on their ability to inhibit the in vitro growth of CML or AML progenitor cells, to be used for the generation of leukemia-reactive CTL lines for clinical use.     

Transient RNA interference in hematopoietic progenitors with functional consequences

Short interfering (si) RNAs have now been shown to inhibit gene expression in several species, including mammals (Elbashir et al.: Nature 411:494-498, 2001; Fire et al.: Nature 391:806-811, 1998). RNA inhibition in primary cells such as stem cells would facilitate rapid gene discovery in a postgenome era. While retroviruses can deliver siRNA expression cassettes for stable expression (Barton and Medzhitov: Proc Natl Acad Sci USA 99:14943-14945, 2002; Paddison et al.: Proc Natl Acad Sci USA 99:1443-1448, 2002; Rubinson et al.: Nat Genet 33:401-406, 2003), an efficient method for direct transfer of siRNA to stem cells is still lacking. Here, we established electroporation to deliver siRNA to hematopoietic progenitors. On average, at least 80% of cells take up the RNA, and these display nearly 100% knockout of marker gene expression at both the RNA and protein level. Moreover, knockdown of the hematopoietic regulator, CD45, results in 3-fold more hematopoietic colonies in a progenitor assay. These results demonstrate that transient transfection of siRNA to primary cells can have substantial functional consequences. This technology may be applicable to a variety of primary cell types.     

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.     

Stem cells remember their grade

The stem cell state is understood based on what cells do in performance assays, crude measures of a highly refined state. In this issue of Cell Stem Cell, Dykstra et al. (2007) reveal stem cell gradation and the extent to which that gradation is retained in stem cell daughters of hematopoietic stem cells.     

The potential of stem cells for the treatment of brain tumors and globoid cell leukodystrophy

Stem cells of different origin are under careful scrutiny as potential new tools for the treatment of several neurological diseases. The major focus of these reaserches have been neurodegenerative disorders, such as Huntington Chorea or Parkinson Disease (Shihabuddin et al., 1999). More recently attention has been devoted to their use for brain repair after stroke (Savitz et al., 2002). In this review we will focus on the potential of stem cell treatments for glioblastoma multiforme (Holland, 2000), the most aggressive primary brain tumor, and globoid cell leukodystrophy (Krabbe disease), a metabolic disorder of the white matter (Berger et al., 2001). These two diseases may offer a paradigm of what the stem cell approach may offer in term of treatment, alone or in combination with other therapeutic approaches. Two kinds of stem cells will be consideredhere: neural stem cells and hematopoietic stem cells, both obtained after birth. The review will focus on experimental models, with an eye on clinical perspectives. This revised version was published online in July 2006 with corrections to the Cover Date.     

Disrupting the stem cell niche: good seeds in bad soil

Stem cells reside in a microenvironment or niche that is critical for stem cell maintenance and regulation. But what happens when a stem cell niche is disrupted? In this issue of Cell, two reports (Walkley et al., 2007a, 2007b) demonstrate in mice that alterations in the niche for hematopoietic stem cells lead to the development of myeloproliferative disease.     

Engineering stem cell expansion

In this issue of Cell Stem Cell, Csaszar et al. (2012) develop a culture method that overcomes current limitations in ex vivo hematopoietic stem/progenitor cell expansion by continuously diluting inhibitory signaling factors and maintaining stem cell density. This approach enhances the generation of precursors with potential therapeutic utility.     

FOXO3-engineered human mesenchymal progenitor cells efficiently promote cardiac repair after myocardial infarction

Dear Editor,Myocardial infarction(MI)is the irreversible cardiomyocyte death resulting from prolonged oxygen deprivation due to obstructed blood supply(ischemia),leading to contractile dysfunction and cardiac remodeling.In recent decades,stem cell transplantation has been extensively investigated for the repair of injured heart in animal studies and clinical trials(Kanelidis et al.,2017;Gyongyosi et al.,2018).     

Uncapping the potential of renal stem cells

In this issue of Cell Stem Cell, McMahon and coworkers (Kobayashi et al., 2008) characterize a subset of progenitor cells in the developing kidney. six2-expressing mesenchymal cells exhibit hallmark stem cell traits, in that they appear to self-renew and are clonally multipotent for a range of nephron epithelial cell types.     

A quantitative assay for insulin-expressing colony-forming progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay. To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 μL of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin βB, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony. In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.     

Development of a novel beta-cell specific promoter system for the identification of insulin-producing cells in in vitro cell cultures

Recently, it has been reported that islet transplantation into patients with Type 1 diabetes may achieve insulin independence for a year or longer [Shapiro et al., Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen, N Engl J Med. 343 (2000) 230-238]. However, the amount of donor islet tissue is limited, therefore, multiple approaches are being explored to generate insulin-producing cells in vitro. Some promising results have been obtained using mouse and human stem cells and progenitor cells [Soria et al., From stem cells to beta cells: new strategies in cell therapy of diabetes mellitus, Diabetologia. 4 (2001) 407-415; Lechner et al., Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus, Am J Physiol Endocrinol Metab. 284 (2003) 259-266; Bonner-Weir et al., In vitro cultivation of human islets from expanded ductal tissue, Proc Natl Acad Sci U S A, 97 (2000) 7999-8004; Assady et al., Insulin production by human embryonic stem cells, 50 (2001) Diabetes 1691-1697]. However, the efficiency of obtaining populations with high numbers of differentiated cells has been poor. In order to improve the efficiency of producing and selecting insulin-producing cells from undifferentiated cells, we have designed a novel beta-cell specific and glucose responsive promoter system designated pGL3.hINS-363 3x. This artificial promoter system exhibits significant luciferase activity not only in insulin-producing MIN6 m9 cells but also in isolated human islets. The pGL3.hINS-363 3x construct shows no activity in non-insulin-producing cells in low glucose conditions (2 mM glucose) but demonstrates significant activity and beta-cell specificity in high glucose conditions (16 mM glucose). Furthermore, pGL3.hINS-363 3x shows significant promoter activity in differentiated AR42J cells that can produce insulin after activin A and betacellulin treatment. Here, we describe a novel beta-cell specific and glucose responsive artificial promoter system designed for analyzing and sorting beta-like insulin-producing cells that have differentiated from stem cells or other progenitor cells.     

Single-cell RNA-Seq analysis identified kidney progenitor cells from human urine

Dear Editor, Urine passes through the entire kidney and urinary tract system starting from the glomerulus and ending to the urethra.Cells in the kidney and urinary tract could be exfoliated from the epithelium into the urine, while leukocyte could infiltrate from the local tissue into the urine, which makes the urine a useful subject for clinical evaluation of relevant diseases.Among them, renal tubular cells and podocytes have been identified and 2D or 3D cultured from human urine specimens (Oliveira Arcolino et al., 2015;Schutgens et al.,2019).Particularly, kidney stem cell/progenitor cells were successfully recovered from pediatric patient urine and then cultured for kidney regenerative purpose by the Romagnani group.However, they also showed that such cells cannot be recovered from healthy individuals (Lazzeri et al., 2015).It remains unknown whether similar types of progenitor cells can be found in different individuals, either healthy or diseased.     

Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells

The ex vivo expansion of hematopoietic progenitor cells is of great interest for a variety of clinical applications, e.g. bone marrow transplantation or gene therapy. Therefore it is of general interest to develop a culture system, able to mimic the in vivo hematopoesis, which is a prerequisite for long-term hematopoietic culture. Our approach was to modify a continuously perfused bioreactor for cultivation and expansion of human hematopoietic stem cells. Therefore we immobilized stromal cells (human primary stromal cells or the murine cell line M2-10B4) in porous glass carriers in a fixed bed reactor and cocultivated human hematopoietic progenitor cells for several weeks. After inoculation of mononuclear cells derived from umbilical cord blood or peripheral blood stem cells both adherent and non adherent cells were harvested and analyzed by flow cytometry and short-term colony assays. During cultivation there was a permanent production of progenitor cells and mature blood cells derived from the immobilized cells in the carriers. We could demonstrate the immobilization of hematopoietic progenitor cells of the myeloid system detectable in short-term colony assays. Additionally we could observe the expansion of very early progenitor cells (CFU-GEMM) up to 4.2-fold and later progenitor cells (CFU-GM and BFU-E) up to 7-fold and 1.8-fold, respectively. P.M. and B.S. contributed equal parts to this work. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential Sensitivity of Mouse Hematopoietic Stem Cells to Merocyanine 540

In vivo and in vitro clonal assays of immature mouse blood cells showed that diffferent populations of hematopoietic progenitor cells differ considerably with respect to their sensitivity to photodynamic damages caused by the fluorescent dye Merocyanine 540. Late erythroid progenitors were the most sensitive cells followed in order of decreasing sensitivity by pluripotent stem cells, early erythroid progenitors, and granulocyte/macrophage progenitors. Only about 2%–4% of all nucleated marrow cells were stained with Merocyanine 540 which correlated well with current frequency estimates of progenitor cells in mouse bone marrow. Our findings indicate that the expression of Merocyanine binding sites is developmentally regulated and might, therefore, provide a useful molecular marker for blood cell differentiation and a basis for an effective purification of hematopoietic progenitor cells.     

Greasing the way: the ABCs of HSPC efflux from the marrow

In this issue of Cell Stem Cell, Westerterp et?al. demonstrate that cholesterol flux through splenic phagocytes regulates hematopoietic stem and multipotential progenitor cell mobilization from the bone marrow. These exciting findings suggest new avenues for therapy of myeloproliferative disorders and atherosclerosis.     

Defining the pathways of early adult hematopoiesis

The journeys that hematopoietic cells take to differentiate from long-term stem cells into committed cells have recently been a topic of debate. In this issue of Cell Stem Cell, Arinobu et al. (2007) and Pronk et al. (2007) provide new insights into the paths traveled by hematopoietic progenitors.     

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.