Stem Cell Kinetics Research Articles

Homeostatic Mass Control in Gastric Non-Neoplastic Epithelia under Infection of Helicobacter pylori: An Immunohistochemical Analysis of Cell Growth, Stem Cells and Programmed Cell Death

We evaluated homeostatic mass control in non-neoplastic gastric epithelia under Helicobacter pylori (HP) infection in the macroscopically normal-appearing mucosa resected from the stomach with gastric cancer, immunohistochemically analyzing the proliferation, kinetics of stem cells and programmed cell death occurring in them. Ki67 antigen-positive proliferating cells were found dominantly in the elongated neck portion, sparsely in the fundic areas and sporadically in the stroma with chronic infiltrates. CD117 could monitor the kinetics of gastric stem cells and showed its expression in two stages of gastric epithelial differentiation, namely, in transient cells from the gastric epithelial stem cells to the foveolar and glandular cells in the neck portion and in what are apparently progenitor cells from the gastric stem cells in the stroma among the infiltrates. Most of the nuclei were positive for ssDNA in the almost normal mucosa, suggesting DNA damage. Cleaved caspase-3-positive foveolar cells were noted under the surface, suggesting the suppression of apoptosis in the surface foveolar cells. Besides such apoptosis of the foveolar cells, in the severely inflamed mucosa apoptotic cells were found in the neck portion where most of the cells were Ki67 antigen-positive proliferating cells. Beclin-1 was recognized in the cytoplasm and in a few nuclei of the fundic glandular cells, suggesting their autophagic cell death and mutated beclin-1 in the nuclei. Taken together, the direct and indirect effects of HP infection on the gastric epithelial proliferation, differentiation and programmed cell death suggested the in-situ occurrence of gastric cancer under HP infection.

Hematopoietic Progenitors with Slow Divisional Kinetics Give Rise to T Cell Precursors in the Thymus of the SCID Mouse Transplantation Model and Represent the Subset with Primitive Function.

Introduction. We have shown in the past that slow divisional kinetics of hematopoietic stem cells (HSC) are correlated with prolonged self renewal and increased frequency of long term culture initiating cells (LTC-IC) and myeloid-lymphoid initiating cells (ML-IC) in vitro. In the present study we have demonstrated that only the slow dividing fraction (SDF) of human CD34+ HSC, but not the fast dividing fraction (FDF), is able to engraft in the thymus and lymph nodes of NOD/SCID mice.Methods. Human CD34+ HSC were isolated from umbilical cord blood, stained with the membrane fluorescence marker PKH26, cultivated in cytokine rich media for five days and then sorted for their PKH26 fluorescence (dim versus bright). Sorted cells were injected into the tail vein of sublethally irradiated NOD/SCID mice (2×10e5 cells / animal). Mice were sacrificed at defined time points from week 2 to week 18 after transplantation.Results. Flow cytometric analysis of the samples collected from the marrow, spleen, thymus, and lymph nodes of the NOD/SCID mice indicated that both SDF and FDF were able to engraft in the bone marrow of the mice, establishing a hematopoietic system, i.e. erythroid and myeloid progenitor cells were recovered from the bone marrow of SDF and FDF reconstituted mice. The most remarkable finding was that only progeny cells of SDF were found in the mouse lymph nodes and thymus where, beginning approximately at week 15, they differentiated into T cells. CD4+CD8+ double positive T progenitor cells were recovered in the thymus and CD4+ as well as CD8+ T cells were present in the spleen and the lymph nodes. However, at least until 18 weeks after reconstitution, very few B cells were present in the NOD/SCID spleen, whereas monocytes reached close to normal values. We are in the process of evaluating the functionality of the human T cells that matured in the NOD/SCID mouse environment. Global gene expression profiles of the two subpopulations (SDF and FDF) were analyzed using a ‘human transcriptome cDNA microarray'. We found that several molecular markers for stem cells were highly expressed in the SDF as compared to FDF: CD133 (Prominin), MDR1 (multiple drug resistance gene 1), clqr1 (complement component 1 receptor 1), Hoxa9, Cdx1 and Hesx1 (which encode homeodomain proteins).Conclusion. Our data indicated that only SDF was enriched for primitive HSC, which in turn were able to engraft in the thymus and lymph nodes in the SCID mouse transplantation model. These cells were able to give rise to T cell precursors and mature T cells. This observation has provided unequivocal evidence that the SDF is associated with primitive HSC function, and is consistent with the results of differential gene expression analysis in SDF versus FDF. The significance and nature of the cell-cell contacts in the microenvironment of the SCID mouse model is currently being examined.

Hypoxia alters progression of the erythroid program

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

Antidepressant drugs reverse the loss of adult neural stem cells following chronic stress

In rodents, adult neurogenesis occurs in the olfactory bulb and the dentate gyrus of the hippocampus. It has been shown that exposure to psychosocial stress reduces cell proliferation in the dentate gyrus. However, little is known about how stress affects the proliferation kinetics of neural stem cells (NSCs) in the subventricular zone (SVZ), which provide new neurons to the olfactory bulb. We utilized a forced-swim model of stress in the mouse and found that chronic stress decreased the number of NSCs in the SVZ. The reduction of NSC number persisted for weeks after the cessation of stress but was reversed by treatment with the antidepressant drugs fluoxetine and imipramine. We demonstrated by in vitro colony-forming neurosphere assay that corticosterone attenuated neurosphere formation by adult NSCs and, in contrast, that serotonin increased the survival of NSCs. In addition, serotonin expanded the size of the NSC pool in the SVZ when it was infused into the lateral ventricle in vivo. These results suggest that, under chronic stress conditions, the number of NSCs is regulated by the actions of glucocorticoids and serotonin. These data provide insights into the molecular mechanisms underlying the pharmacological actions of antidepressant drugs.

HIV enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in HIV/Microsporidia-infected jejunal mucosa

The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.

Clonal analysis of thymus-repopulating cells presents direct evidence for self-renewal division of human hematopoietic stem cells

AbstractTo elucidate the in vivo kinetics of human hematopoietic stem cells (HSCs), CD34+CD38– cells were infected with lentivirus vector and transplanted into immunodeficient mice. We analyzed the multilineage differentiation and self-renewal abilities of individual thymus-repopulating clones in primary recipients, and their descending clones in paired secondary recipients, by tracing lentivirus gene integration sites in each lymphomyeloid progeny using a linear amplification-mediated polymerase chain reaction (PCR) strategy. Our clonal analysis revealed that a single human thymus-repopulating cell had the ability to produce lymphoid and myeloid lineage cells in the primary recipient and each secondary recipient, indicating that individual human HSCs expand clonally by self-renewal division. Furthermore, we found that the proportion of HSC clones present in the CD34+ cell population decreased as HSCs replicated during extensive repopulation and also as the differentiation capacity of the HSC clones became limited. This indicates the restriction of the ability of individual HSCs despite the expansion of total HSC population. We also demonstrated that the extensive self-renewal potential was confined in the relatively small proportion of HSC clones. We conclude that our clonal tracking studies clearly demonstrated that heterogeneity in the self-renewal capacity of HSC clones underlies the differences in clonal longevity in the CD34+ stem cell pool.

Kinetics of progenitor hemopoetic stem cells in sepsis: Correlation with patients survival?

BackgroundCurrent theories underline the crucial role of pro-inflammatory mediators produced by monocytes for the pathogenesis of sepsis. Since monocytes derive from progenitor hemopoetic cells, the kinetics of stem cells was studied in peripheral blood of patients with sepsis.MethodsBlood was sampled from 44 patients with septic syndrome due to ventilator-associated pneumonia on days 1, 3, 5 and 7 upon initiation of symptoms. Concentrations of tumour necrosis factor-alpha (TNFα), interleukin (IL)-6, IL-8 and G-CSF were estimated by ELISA. CD34/CD45 cells were determined after incubation with anti-CD45 FITC and anti-CD34 PE monocloncal antibodies and flow cytometric analysis. Samples from eight healthy volunteers served as controls.ResultsMedian of CD34/CD45 absolute count of controls was 1.0/μl. Respective values of the total study population were 123.4, 112.4, 121.5 and 120.9/μl on days 1, 3, 5 and 7 (p < 0.0001 compared to controls). Positive correlations were found between the absolute CD34/CD45 count and the absolute monocyte count on days 1, 5 and 7. Survival was prolonged among patients with less than 310/μl CD34/CD45 cells on day 1 compared to those with more than 310/μl of CD34/CD45 cells (p: 0.022). Hazard ratio for death due to sepsis was 5.47 (p: 0.039) for CD34/CD45 cells more than 310/μl. Median IL-6 on day 1 was 56.78 and 233.85 pg/ml respectively for patients with less than 310/μl and more than 310/μl CD34/CD45 cells (p: 0.021).ConclusionStem cells are increased in peripheral blood over all days of follow-up compared to healthy volunteers. Patients with counts on day 1 less than 310/μl are accompanied by increased survival compared to patients with more than 310/μl.

Bayesian inference in a hidden stochastic two-compartment model for feline hematopoiesis

In this paper, we describe a hidden two-compartment stochastic process used to model the kinetics of feline hematopoietic stem cells (HSCs) in continuous time. Because of the experimental design and data collection scheme, the inferential task presents numerous challenges. While the hematopoietic process evolves in continuous time, the observations are collected only at discrete irregular times and are a probabilistic function of the state of the process. In addition, the animals go through an experimental procedure such that their reserve of HSCs is severely depleted at the start of the observation period. This impedes any approximation of the hematopoietic process with a continuous state-space process (normal approximation of the transition probabilities would be inaccurate when the state of the process, i.e. the number of stem cells, is small). We implement a Markov chain Monte Carlo algorithm that allows us to estimate the posterior distribution of the parameters of the hematopoietic process while maintaining its state-space discrete (i.e. without using any approximation). We show the performance of the algorithm on simulated data. Finally, we apply the algorithm to data on multiple experimental cats and provide estimates of the rates of the fates of feline HSCs. The obtained estimates are in agreement with the estimates obtained with different methods published in the medical literature. However, the proposed approach makes a more efficient use of the data and hence the parameter estimates are much more accurate than the one obtained with the methods previously proposed.

Isolation and Characterization of a Putative Intestinal Stem Cell Fraction From Mouse Jejunum

Although there have been many recent advances regarding the biology of intestinal stem cells, the field has been hampered significantly by the lack of a method to isolate these cells. Therefore, the aim of this study was to explore the hypothesis that viable intestinal stem cells can be isolated as a side population (SP) by fluorescence-activated cell sorting after staining with the DNA-binding dye Hoechst 33342. Preparations of individual cells from either whole mucosa or epithelium of mouse jejunum were stained with Hoechst 33342 and propidium iodide and then sorted using fluorescence-activated cell sorting. Cells were characterized using fluorochrome-labeled antibodies to surface markers, intracellular markers, and annexin V to detect early apoptosis. Total RNA was isolated from sorted fractions and used for quantitative real-time reverse-transcription polymerase chain reaction to evaluate the expression of cell lineage markers and the intestinal stem-cell marker, Musashi-1. Adult and neonatal jejunum contain a viable population of cells that shows the SP phenotype and is sensitive to verapamil. This population of cells (from both mucosal and epithelial preparations) includes a CD45-negative fraction corresponding to nonhematopoietic cells, which shows minimal expression of surface markers typically found on stem cells from other tissues and of intracellular markers found in mesenchymal cells. Additionally, these cells were enriched for Musashi-1 and beta1-integrin, were cytokeratin positive, and survived in culture for up to 14 days. The CD45-negative SP fraction, although not pure, represents the successful isolation of a viable population significantly enriched in small intestinal epithelial stem cells.

The kinetics of clonal dominance in myeloproliferative disorders

AbstractTo study clonal evolution in myeloproliferative disorders, we used stochastic models of hematopoiesis for mouse and cat, species for which the in vivo kinetics of hematopoietic stem cells (HSCs) have been experimentally defined. We determined the consequence if 1 HSC became able to survive without the support of a microenvironmental niche while the rest of its behavior did not change. Neoplastic cells persisted and dominated hematopoiesis in 14% of mice and 17% of cats, requiring mean times of 2.5 ± 0.5 and 7.0 ± 1.2 years, respectively (n = 1000 simulations/species). In both species, when the number of neoplastic HSCs exceeded 0.5% of all HSCs, clonal dominance was inevitable. Our results can explain the absence of clonal myeloproliferative disorders in mice (lifetime, 2 years), are consistent with clinical observations in cats, and provide insight into the progression of chronic myelogenous leukemia (CML) in humans. They also demonstrate that competition for microenvironmental support can lead to the suppression of normal hematopoiesis as neoplasia evolves. Toxic or immunologic suppression of normal HSCs is not required.

Promotion of Cardiac Regeneration by Cardiac Stem Cells

See related article, pages 663–673 Research on myocardial regeneration is an exciting and promising area, which challenges the dogma that the heart is a nonregenerating organ. Recently, several methods of stem cell therapy have been developed. One method is to transplant cells into the infarcted area of the myocardium. Currently, clinical trials of autologous skeletal myoblast transplantation into the failed heart are underway and have been reported to improve the cardiac function.1 However, the mechanism of its efficacy is unknown, and there are some questions about the safety because myoblasts do not transdifferentiate into cardiomyocytes and may induce lethal arrhythmia.1 In this point, embryonic stem (ES) cells that can differentiate into cardiomyocytes are thought to be more promising.2 For patients experiencing extensive myocardial infarction or dilated cardiomyopathy, however, the effectiveness of cell transplantation is questionable. Bone marrow-derived cells have been reported to transdifferentiate into various types of cells in situ. Indeed, bone marrow-derived stem cells were reported to prevent left ventricular remodeling after myocardial infarction and improve cardiac function by their differentiation into cardiomyocytes.3 However, recent accumulating evidence has indicated that very few bone marrow cells, if any, transdifferentiate into cardiomyocytes.4–6 Cytokine therapy using G-CSF strongly prevents ventricular remodeling after myocardial infarction by antiapoptotic and angiogenic effects, but not by recruitment of bone marrow cells.7 Over the past few years, adult hearts have been reported to contain the cardiac stem/progenitor cells such as c-kit+,8 Sca-1+,9,10 isl-1+,11 and side population cells.12 Because these cells have the ability to proliferate and …

Chronic myeloid leukaemia: stem cell derived but progenitor cell driven

The biology of CML (chronic myeloid leukaemia) has been extensively investigated as the disease is a paradigm of neoplasms induced when a translocation results in expression of a novel fusion protein, in this instance p210(BCR-ABL). Although CML manifests itself principally as unregulated expansion of the myeloid lineage, the lesion is present in the stem cell population and it has long been assumed that disregulated stem cell kinetics must underlie the basic pathology of the disease. In this review, we present evidence that, in normal haemopoiesis, less primitive precursor cells retain considerable flexibility in their capacity to undergo self-renewal, allowing them to maintain lineage-specific homoeostasis without inflicting proliferative stress upon the stem cell population. This mechanism is dysregulated in CML and we have developed a self-renewal assay for CFU-GM (colony-forming unit-granulocyte/macrophage) which demonstrates that, in CML, the PI (proliferative index) of the myeloid progenitor cell population is increased. The ability to measure the PI as an endpoint of p210(BCR-ABL) expression gives considerable versatility to the in vitro investigation of putative therapeutic regimes in CML.

Engraftment Kinetics of Human Cord Blood and Murine Fetal Liver Stem Cells Following In Utero Transplantation into Immunodeficient Mice

This study was undertaken to evaluate the kinetics of engraftment after in utero transplantation of murine fetal liver and human cord blood stem cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. NOD/SCID fetuses were injected with murine fetal liver or human cord blood CD34+ cells at day 13.5 of gestation. Frequencies of donor cells were analyzed by flow cytometry up to 48 h post transplantation and 4-16 weeks postnatally. Hematopoietic multilineage reconstitution capacity was assessed. Both types of donor cells home rapidly. However, the frequency of human cord blood stem cells rapidly diminished while the murine fetal liver stem cells expanded over time, resulting in multilineage hematopoietic reconstitution. Differences in long-term reconstitution of allogeneic versus xenogeneic donor cells were ascribed to the inability of the human cells to self-renew and differentiate in the fetal mouse environment, demonstrating the limitations of this commonly used xenograph.

Growth Kinetics of Human Mesenchymal Stem Cells from Bone Marrow and Umbilical Cord Blood

Growth Kinetics of Human Mesenchymal Stem Cells from Bone Marrow and Umbilical Cord Blood

Quiescent (5-fluorouracil-resistant) aplastic anemia hematopoietic cells in vitro

Objective Bone marrow from aplastic anemia (AA) patients shows reduced numbers in long-term culture (LTC)-initiating cell (LTC-IC) assays. The LTC-IC assay is based on assumptions of the culture kinetics of normal hematopoietic stem cells (HSC), which are not necessarily justified in a disease state. We therefore undertook a detailed examination of the kinetics of quiescent HSC from AA patients in LTC. Methods Colony formation by quiescent HSC in LTC was tested by pretreating control (n = 6) and AA bone marrow (n = 7) with 5-fluorouracil. Secondly, we manipulated normal samples to inoculate cultures with proportions of CD34 + cells similar to those from AA samples. We obtained enough CD34 + cells to reconstitute one AA sample to “normal” levels. Results Patient cells showed altered kinetics with rapid proliferation and premature termination of LTC. In vivo, decreased numbers of HSC may induce rapid proliferation and differentiation; a similar phenomenon could explain the observations in culture. We therefore manipulated normal samples to contain a proportion of CD34 + HSC similar to that in AA samples. Although absolute numbers of secondary colonies in LTC were reduced, the kinetics of culture were not altered. However, when AA CD34 + HSC were reconstituted to “normal” levels, the cultures still demonstrated early termination. Conclusions The kinetics of LTC are not affected by CD34 + HSC number. However, quiescent HSC derived from patients with AA have qualitative differences from normal cells, as reflected by distinct kinetics in long-term culture. This has implications for the interpretation of the LTC-IC assay with AA samples.

Hematopoiesis as a competitive exclusion process: Estimation of a stem cell selective advantage

Female Safari cats are offspring of domestic and Geoffroy parents, and are balanced heterozygotes with equal numbers of Blood cells containing domestic and Geoffroy-type glucose 6-phosphate dehydrogenase (G6PD),an X-chromosome derived enzyme. In previous studies, however, we observed increasing percentages of Blood cells with Geoffroy G6PD in cats aged 4–12 years. Similarly, ratios of parental X-chromosome phenotypes greater than three to one are common in women over age 60, while women under 40 typically exhibit one to one ratios. Using a two-compartment hidden-Markov model of the distribution of phenotype in samples taken during the second stage of hematopoiesis (Blood cell production), we estimate, through simulation and mathematical calculation, the Geoffroy selective advantages necessary to reproduce the data obtained from female Safari cats. It is shown that small differences in the kinetics of hematopoietic stem cells (HSC), less than might be detected by in vitro assays, can explain the “clonal dominance” observed with aging in cats, and by extension, human females. Conceptually, hematopoiesis in females can be viewed as a competitive exclusion process in which two populations of HSC (defined by parental X-chromosome phenotype) compete for environmental resources. As in many ecological examples, dominance may occur only after long periods of time.

In vivo self-renewing divisions of haematopoietic stem cells are increased in the absence of the early G1-phase inhibitor, p18INK4C.

Self-renewal of stem cells is critical for tissue repair and maintenance of organ integrity in most mammalian systems. The relative asymmetry between self-renewal and differentiation in balance with apoptosis determines the size and durability of a stem-cell pool. Regulation of the cell cycle is one of the fundamental mechanisms underlying determination of cell fate. Absence of p21(Cip1/Waf1), a late G1-phase cyclin-dependent kinase inhibitor (CKI), has previously been shown to enable cell-cycle entry of haematopoietic stem cells, but leads to premature exhaustion of the stem cells under conditions of stress. We show here that deletion of an early G1-phase CKI, p18(INK4C), results in strikingly improved long-term engraftment, largely by increasing self-renewing divisions of the primitive cells in murine transplant models. Therefore, different CKIs have highly distinct effects on the kinetics of stem cells, possibly because of their active position in the cell cycle, and p18(INK4C) appears to be a strong inhibitor limiting the potential of stem-cell self-renewal in vivo.

256.Development of a label retaining cell method to identify stem cells in mouse endometrium

Epithelial and stromal stem/progenitor cells have been demonstrated in human endometrium by their clonogenic activity (1). To understand the regulation and growth kinetics of endometrial stem cells (SCs), an animal model is being established using the label retaining cell (LRC) approach. Tissue SCs are quiescent and will retain a DNA synthesis label (BrdU), while the label is diluted out in more mature dividing cells during a chase period. Those cells retaining the label over long period of time are LRCs and have been identified as SCs (2). An optimum time to initially label the majority of the cells is critical. We hypothesised that during mouse endometrial development, there is a growth window for maximum labelling of epithelial and stromal cells. The aim of this study was to determine the optimum age and length of time for maximum endometrial BrdU labelling. Two stages of endometrial growth were exploited: (1) postnatal growth before gland development (3 days old – P3); and (2) oestrogen induced growth in ovariectomised prepubertal mice (4–5 weeks old). C57/CBA P3 mice received subcutaneous BrdU injections twice daily for 3 consecutive days. Four to five-week-old ovariectomised mice received BrdU filled mini-osmotic pumps implanted subcutaneously for 7 and 14 days, with daily oestrogen injections. Mice were killed 4 h after the last treatment, uteri collected, and BrdU labelled cells were detected by immunohistochemistry. Results are shown in the table below (see PDF file). The data shows that maximum labelling for epithelial and stromal cells was achieved for P3 mice, possibly due to greater endometrial growth of the Müllerian duct compared to oestrogen induced endometrial growth following ovariectomy. Chase experiments are currently in progress on P3 labelled mice to identify the location of LRCs in mouse endometrial glands and stroma. (1) Chan RW, Schwab KE, Gargett CE (2004) Biol. Reprod. 70, in press. (2) Morris RJ, Potten CS (1994) Cell Prolif. 27, 279–289.

Modeling stem cell population growth: incorporating terms for proliferative heterogeneity.

Expansion of the undifferentiated stem cell phenotype is one of the most challenging aspects in stem cell research. Clinical protocols for stem cell therapeutics will require standardization of defined culture conditions. A first step in the development of predictable and reproducible, scalable bioreactor processes is the development of mathematical growth models. This paper provides practical models for describing cell growth in general, which are particularly well suited for examining stem cell populations. The nonexponential kinetics of stem cells derive from proliferative heterogeneity, which is biologically recognized as mitosis, quiescence, senescence, differentiation, or death. Here, we examined the assumptions of the Sherley model, which describes heterogeneous expansion in the absence of cell loss. We next incorporated terms into the model to account for A) cell loss or apoptosis and B) cell differentiation. We conclude that the basic assumptions of the model are valid and a high correlation between the modified equations and experimental data obtained using muscle-derived stem cells was observed. Finally, we demonstrate an improved estimation of the kinetic parameters. This study contributes to both the biological and mathematical understanding of stem cell dynamics. Further, it is expected that the models will prove useful in establishing standardization of cell culture conditions and scalable systems and will be required to develop clinical protocols for stem cell therapeutics.

Homing efficiency and proliferation kinetics of male germ line stem cells following transplantation in mice.

Stem cells in the male germ line (spermatogonial stem cells [SSCs]) are an important target for male fertility restoration and germ line gene modification. To establish a model system to study the biology and the applications of SSCs in mice, I used a sequential transplantation strategy to analyze the process by which SSCs colonize the stem cell niche after transplantation and to determine the efficiency of the process (homing efficiency). I further analyzed the proliferation kinetics of SSCs after colonization. The number of SSCs gradually decreased during the homing process, and only 12% of SSCs successfully colonized the niche on Day 7 after transplantation, but the number of SSCs increased by Day 14. Thus, homing efficiency of adult mouse SSCs is 12%. These results indicate that SSCs are rapidly lost upon transplantation and require approximately 1 wk to settle into their niches before initiating expansion. Using this SSC homing efficiency, I calculated that approximately 3000 SSCs exist in one normal adult testis, representing approximately 0.01% of total testis cells. Between 7 days and 1 mo after transplantation, SSCs proliferated 7.5-fold. However, they did not significantly proliferate thereafter until 2 mo, and only 8 SSCs supported one colony of donor-derived spermatogenesis from 1 to 2 mo. These results suggest that self-renewal and differentiation of SSCs are strictly regulated in coordination with the progress of an entire unit of regenerating spermatogenesis.