Cell Self-renewal Ability Research Articles

Inherited myogenic abilities in muscle precursor cells defined by the mitochondrial complex I-encoding protein

Skeletal muscle comprises different muscle fibers, including slow- and fast-type muscles, and satellite cells (SCs), which exist in individual muscle fibers and possess different myogenic properties. Previously, we reported that myoblasts (MBs) from slow-type enriched soleus (SOL) had a high potential to self-renew compared with cells derived from fast-type enriched tibialis anterior (TA). However, whether the functionality of myogenic cells in adult muscles is attributed to the muscle fiber in which they reside and whether the characteristics of myogenic cells derived from slow- and fast-type fibers can be distinguished at the genetic level remain unknown. Global gene expression analysis revealed that the myogenic potential of MBs was independent of the muscle fiber type they reside in but dependent on the region of muscles they are derived from. Thus, in this study, proteomic analysis was conducted to clarify the molecular differences between MBs derived from TA and SOL. NADH dehydrogenase (ubiquinone) iron-sulfur protein 8 (Ndufs8), a subunit of NADH dehydrogenase in mitochondrial complex I, significantly increased in SOL-derived MBs compared with that in TA-derived cells. Moreover, the expression level of Ndufs8 in MBs significantly decreased with age. Gain- and loss-of-function experiments revealed that Ndufs8 expression in MBs promoted differentiation, self-renewal, and apoptosis resistance. In particular, Ndufs8 suppression in MBs increased p53 acetylation, followed by a decline in NAD/NADH ratio. Nicotinamide mononucleotide treatment, which restores the intracellular NAD+ level, could decrease p53 acetylation and increase myogenic cell self-renewal ability in vivo. These results suggested that the functional differences in MBs derived from SOL and TA governed by the mitochondrial complex I-encoding gene reflect the magnitude of the decline in SC number observed with aging, indicating that the replenishment of NAD+ is a possible approach for improving impaired cellular functions caused by aging or diseases.

KIF15 knockdown inhibits the development of endometrial cancer by suppressing epithelial-mesenchymal transition and stemness through Wnt/β-catenin signaling.

Endometrial cancer (EC) is one of the most common cancers among women, while the incidence of EC is rising. Many studies have found that Kinesin family member 15 (KIF15) is highly expressed in a series of cancers, but the role of KIF15 in EC is unclear. We detected the expression level of KIF15 in a microarray of EC tissues by immunohistochemical staining (IHC), and analyzed the correlation between the expression level of KIF15 and the pathological characteristics of patients. After inhibit the expression of KIF15 in EC cells with lentivirus, cell proliferation and apoptosis were detected respectively by CCK8 assay, flow cytometry and tunnel assay. Transwell assay and wound healing assay were used to examine the migration ability and invasion ability of EC cells. Spheroid formation assay was used to evaluate cell self-renewal ability. In vivo tumor xenograft model was used for validation. The expressions of epithelial-mesenchymal transition, cancer stem cells, and Wnt/β-catenin signaling molecules were detected by Western blotting. The results showed that the expression of KIF15 in EC tissues was higher than that in normal endometrial tissues, while the expression level of KIF15 in EC was positively correlated with the pathological grade of the tumor. The down-regulation of KIF15 reduced the proliferation, colony formation, invasion, migration and self-renewal ability of EC cells, while promoted cell apoptosis. Knockdown of KIF15 inactivates the Wnt/β-catenin signaling of EC cells, inhibitors of Wnt signaling can counteract the enhanced self-renewal ability caused by KIF15 overexpression. Therefore, KIF15 may be a new potential target for diagnosis and treatment of EC.

TBX3 stimulates proliferation and stem cell self-renewal in bladder carcinoma.

With the change of people's lifestyle in recent years, bladder carcinoma has been the second leading cause of death for men. Nevertheless, surgical results of bladder carcinoma are unsatisfying with recurrence and distant metastasis. Therefore, it is urgent to find a new target for bladder carcinoma treatment. The protein and mRNA expression levels of TBX3 in bladder carcinoma tissue samples and cells were tested using western blot and qRT-PCR assays, respectively. Cancer stem cells (CSCs) were separated with immunomagnetic beads. Expression levels of cell stemness-associated proteins CD44, CD24 and ESA in T24 CSCs and T24 cells were detected by western blot assay. Cell self-renewal ability was detected by stem cell sphere formation assay. CCK-8 and colony formation assays examined cell viability and proliferation. Cell apoptotic level was examined by flow cytometry. Elevated TBX3 expression in bladder carcinoma stimulated cell proliferation and inhibited cell apoptosis. Stemness-related proteins and TBX3 were highly expressed in T24 CSCs relative to those in normal bladder carcinoma cells. In addition, TBX3 promoted stem cell self-renewal and inhibited cell apoptosis. Finally, qRT-PCR, western blot and cell sphere formation assays revealed that the potential role of TGF-β1 in the regulation of TBX3. TBX3, mediated by TGF-β1, can promote bladder carcinoma cell proliferation, inhibit apoptosis, and enhance cell stemness. Hence, TBX3 is a potential target to stem cells of bladder carcinoma.

Epigenetic Regulator KDM4D Restricts Tumorigenesis via Modulating SYVN1/HMGB1 Ubiquitination Axis in Esophageal Squamous Cell Carcinoma.

BackgroundIncreasing researches have been reported that epigenetic alterations play critical roles in ESCC development. However, the role of the histone demethylase KDM4D in ESCC tumorigenesis is poorly investigated. This study aims to discover the underlying mechanisms between KDM4D and ESCC progression.MethodsCCK-8 assays, clone formation assay and soft-agar assays were performed to assess cell proliferation. Transwell assay was utilized to assess cell migration efficiency, while sphere formation assay was used to evaluate the cell self-renewal ability. Bioinformatic analysis was conducted to identify prognostic factors and predict the potential E3 ubiquitin ligases. In vitro ubiquitination assay was conducted to confirm the regulations between SYVN1 and HMGB1. The mRNA levels or protein levels of genes were detected by real-time PCR and western blot analysis. In vivo tumor xenograft models were used to determine whether the HMGB1 inhibition affected the malignant features of ESCC cells.ResultEpigenome screening and low-throughput validations highlighted that KDM4D is a tumor suppressor in ESCC. KDM4D expressed lowly in tumors that predicts poor prognosis. KDM4D deficiency significantly enhanced tumor growth, migration and stemness. Mechanistically, KDM4D transcriptionally activates SYVN1 expressions via H3K9me3 demethylation at the promoter region, thereby triggering the ubiquitin-dependent degradation of HMGB1. Low KDM4D depended on accumulated HMGB1 to drive ESCC progression and aggressiveness. Targeting HMGB1 (Glycyrrhizin) could remarkably suppress ESCC tumor growth in vitro and in vivo, especially in KDM4D-deficient cells.ConclusionsWe systematically identified KDM4D/SYVN1/HMGB1 axis in ESCC progression, proving novel biomarkers and potential therapeutic targets.

Differentiation of Human Embryonic Stem Cells into Neuron, Cholinergic, and Glial Cells.

Human embryonic stem cells (hESCs) are pluripotent cells, capable of differentiation into different cellular lineages given the opportunity. Derived from the inner cell mass of blastocysts in early embryonic development, the cell self-renewal ability makes them a great tool for regenerative medicine, and there are different protocols available for maintaining hESCs in their undifferentiated state. In addition, protocols for differentiation into functional human neural stem cells (hNSCs), which have the potential for further differentiation into various neural cell types, are available. However, many protocols are time-consuming and complex and do not always fit for purpose. In this study, we carefully combined, optimized, and developed protocols for differentiation of hESCs into adherent monolayer hNSCs over a short period of time, with the possibility of both expansion and freezing. Moreover, the method details further differentiation into neurons, cholinergic neurons, and glial cells in a simple, single step by step protocol. We performed immunocytochemistry, qPCR, and electrophysiology to examine the expression profile and characteristics of the cells to verify cell lineage. Using presented protocols, the creation of neuronal cultures, cholinergic neurons, and a mixed culture of astrocytes and oligodendrocytes can be completed within a three-week time period.

FLI-06 Intercepts Notch Signaling And Suppresses The Proliferation And Self-renewal Of Tongue Cancer Cells.

PurposeThe Notch signaling pathway plays an oncogenic role in tongue squamous cell carcinoma. The aim of this study was to inhibit the proliferation and self-renewal of tongue cancer cells by applying Notch signaling pathway inhibitor FLI-06 (Selleck, USA) and to lay a foundation for the clinically targeted treatment of tongue cancer for the future.MethodsThe mRNA expression level of Notch1 and the overall survival rate of patients with tongue cancer were examined by analyzing the TCGA database. Tongue cancer cells were treated with FLI-06. Cell proliferation, apoptosis, and stem cell self-renewal ability were tested in appropriate ways. A xenograft mouse model was established to observe tumor growth.ResultsFrom the TCGA data, we demonstrated that patients with high expression of Notch1 had a poor prognosis. We observed that the Notch signaling pathway inhibitor FLI-06 can restrain the activation of the Notch signaling pathway, decrease cell proliferation and induce cell apoptosis in vitro. The xenograft experiment indicated that intraperitoneal injection of FLI-06 inhibited tumor growth and increased cell apoptosis. FLI-06 suppressed both the mRNA and protein expression of Notch receptor and Notch targeted genes. We also observed that FLI-06 suppressed the proliferation of tongue cancer stem cells.ConclusionFLI-06 can block the proliferation and self-renewal of tongue cancer cells. It is inferred that this compound, which inhibits the Notch signaling pathway, may serve as a potential targeted drug for the treatment of tongue cancer in the clinic.

Downregulation of microRNA-196a inhibits stem cell self-renewal ability and stemness in non-small-cell lung cancer through upregulating GPX3 expression.

Studies have reported a high expression profile of microRNA-196a (miR-196a) in many cancers, which potently plays important roles in carcinogenesis. However, the involvement of miR-196a in affecting non-small cell lung cancer (NSCLC) carcinogenesis still remains uncertain. NSCLC-related differentially expressed genes were retrieved for this study according to the microarray-based analysis, which demonstrated that miR-196a may be involved in NSCLC progression via regulation of the Jun N-terminal kinase (JNK) pathway by targeting glutathione peroxidase 3 (GPX3). Hence, this study aimed to explore the relationship among miR-196a, GPX3, and the JNK pathway and to investigate its functional regulations in NSCLC. Initially, highly-expressed miR-196a and lowly-expressed GPX3 were determined in NSCLC tissues and cells. Next, the NSCLC cells were manipulated with a series of mimic, inhibitor or shRNA to investigate the impact of miR-196a and GPX3 on CSC viability, proliferation, self-renewal ability and stemness. The in vivo effect of miR-196a was measured in nude mice xenografted with NSCLC cells. The results demonstrated that downregulation of miR-196a and restoration of GPX3 inhibited CSC viability, proliferation, self-renewal ability, stemness and tumorigenicity. Meanwhile, the underlying relationship among miR-196a, GPX3 and JNK pathway was explored by treatment with the JNK pathway inhibitor (SP600125), or sh-GPX3. Downregulated miR-196a and upregulated GPX3 could elevate the GPX3 protein expression and reduce the extent of JNK and c-Jun phosphorylation. Taken together, miR-196a promotes the development of NSCLC via activation of the JNK pathway through down-regulation of GPX3 and serve as a potential therapeutic target in NSCLC.

Cross-talk among AFAP1-AS1, ACVR1 and microRNA-384 regulates the stemness of pancreatic cancer cells and tumorigenicity in nude mice

BackgroundPancreatic cancer (PC) represents one of the most aggressive forms of cancer. The role of long non-coding RNAs (lncRNAs) has been highlighted in various malignancies including PC. The aim of the present study was to investigate the effects associated with actin filament-associated protein 1 antisense RNA 1 (AFAP1-AS1) on the progression of PC and the underlying mechanism.MethodsMicroarray-based gene expression profiling of PC was performed to identify PC-related lncRNAs, after which the expression of AFAP1-AS1 and cancer stem cell (CSC) markers in PC tissues and cells were determined accordingly. The potential microRNA-384 (miR-384) capable of binding to AFAP1-AS1, in addition to its ability to regulate activin receptor A type I (ACVR1) were analyzed. In order to investigate the effect of the AFAP1-AS1/miR-384/ACVR1 axis on self-renewal ability, tumorigenicity, invasion, migration and stemness of PC cells, shRNA-AFAP1-AS1, miR-384 mimic and inhibitor were cloned into cells.ResultsHigh expression of AFAP1-AS1 and ACVR1 with low expression of miR-384 were detected in PC tissues. ACVR1 was determined to be down-regulated when miR-384 was overexpressed, while the inhibition of AFAP1-AS1 decreased its ability to binding competitively to miR-384, resulting in the down-regulation of ACVR1 and enhancing miR-384 expression, ultimately inhibiting the progression of PC. The knockdown of AFAP1-AS1 or overexpression of miR-384 was confirmed to impair PC cell self-renewal ability, tumorigenicity, invasion, migration and stemness.ConclusionsTaken together, AFAP1-AS1 functions as an endogenous RNA by competitively binding to miR-384 to regulate ACVR1, thus conferring inhibitory effects on PC cell stemness and tumorigenicity.

Maintained Properties of Aged Dental Pulp Stem Cells for Superior Periodontal Tissue Regeneration.

Owing to excellent therapeutic potential, mesenchymal stem cells (MSCs) are gaining increasing popularity with researchers worldwide for applications in tissue engineering, and in treatment of inflammation-related and age-related disorders. However, the senescence of MSCs over passaging has limited their clinical application owing to adverse effect on physiological function maintenance of tissues as well as disease treatment. An inflammatory microenvironment is one of the key contributors to MSC senescence, resulting in low regeneration efficiency. Therefore, MSCs with high resistance to cellular senescence would be a benefit for tissue regeneration. Toward this end, we analyzed the senescence properties of different types of stem cells during culture and under inflammation, including dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), bone marrow mesenchymal stem cells (BMMSCs), and adipose-derived stem cells (ADSCs). Overall, the DPSCs had higher proliferation rates, lower cellular senescence, and enhanced osteogenesis maintenance compared to those of non-dental MSCs cultured from passage three to six. The expression profiles of genes related to apoptosis, cell cycle, and cellular protein metabolic process (contributing to the cell self-renewal ability and metabolic processes) significantly differed between DPSCs and BMMSCs at passage three. Moreover, DPSCs were superior to BMMSCs with regards to resistance to lipopolysaccharide-induced apoptosis and senescence, with enhanced osteogenesis in vitro, and showed improved periodontal regeneration after injection in a miniature pig periodontitis model in vivo. Overall, the present study indicates that DPSCs show superior resistance to subculture and inflammation-induced senescence and would be suitable stem cells for tissue engineering with inflammation.

Abstract 1133: Mitochondria localized EGFRvIII promotes stemness through regulation of mitochondrial dynamics in glioblastoma stem cells

Abstract Purpose Changes in mitochondrial morphology have been linked with mitochondrial function and host cell homeostasis in many metabolic diseases. Cancer is increasingly perceived as metabolic disease, so understanding and regulating mitochondrial dynamics is of interest in cancer therapeutics. Recent studies indicate that distinct metabolic profile of cancer is dependent on genes that regulate the fusion and fission of mitochondria. Importantly, high levels of mitochondrial fission activity are common in cancer cell malignancy and in stem cell′s resistance to differentiation. Since these two phenotypes converge into cancer stem cells, investigating mitochondrial fission activity will provide hints to target cancer stem cell in therapeutics. Purpose of this study is to elucidate what regulates mitochondrial dynamics in cancer stem cells to sustain their stemness properties. In GBM, amplification of the oncogenic variant of EGFR, EGFRvIII, is associated with poor patient prognosis and has recently been known as the major contributor for stemness. Thus, we investigated the role of this oncogenic change in mitochondrial dynamics and stemness control in glioblastoma stem cells (GSCs). Methods Patient-derived GSCs were cultured and sorted by FACS based on tetramethylrhodamine ethyl ester (TMRE) dye emission level, which indicates mitochondrial membrane potential and functional activeness. The highest and the lowest TMRE groups each was collected and subject to further experiments. siRNA was used for loss-of-function study and lentivirus containing overexpression vector was used for gain-of-function study. In order to show the effect of EGFRvIII kinase activity inhibition, gefitinib and its derivative were used. Results High TMRE GSCs showed stronger self-renewal ability in spheroid culture and higher levels of stemness marker Nestin, EGFRvIII, and mitochondrial fission regulator DRP1 expression than low TMRE GSCs. The high TMRE GSCs also showed more fragmented mitochondrial pattern, indicative of active mitochondrial fission. As underlying mechanism of EGFRvIII action on mitochondrial membrane potential, we figured out that EGFRvIII is localized at mitochondria. In order target mitochondria localized EGFRvIII, we used a gefitinib derivative which has mitochondria targeting moiety. This drug reduced mitochondrial membrane potential, self-renewal property, protein levels that regulate mitochondrial dynamics and less fragmented mitochondria, compared to gefitinib treated cells. Conclusion EGFRvIII promotes self-renewal property of GSCs by translocating to mitochondria and upregulating mitochondrial membrane potential and fission-related protein levels. This finding highlights the role of mitochondrial dynamics as a mediator mechanism between tumor-specific oncogenic change and stem cell self-renewal ability. Citation Format: Yeonhee You, Jun Hee Hong, Jinlong Yin, Young Taek Oh, Sung Soo Kim, Jong Bae Park. Mitochondria localized EGFRvIII promotes stemness through regulation of mitochondrial dynamics in glioblastoma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1133.

Myocardial infarction does not affect circulating haematopoietic stem and progenitor cell self-renewal ability in a rat model.

What is the central question of this study? Although peripheral blood haematopoietic stem and progenitor cells are potentially important in regeneration after acute myocardial infarction, their self-renewal ability in the post-acute phase has not yet been addressed. What is the main finding and its importance? In rat peripheral blood, we show that myocardial infarction does not negatively affect circulating haematopoietic stem and progenitor cell self-renewal ability 2 weeks after acute infarction, which suggests a constant regenerative potential in the myocardial infarction post-acute phase. Given the importance of peripheral blood haematopoietic stem and progenitor cells (HPCs) in post-acute regeneration after acute myocardial infarction (MI), the aim of the present study was to investigate the number and secondary replating capacity/self-renewal ability of HPCs in peripheral blood before and 2weeks after MI. In female Lewis inbred rats (n=9), MI was induced by ligation of the left coronary artery, and another nine underwent sham surgery, without ligation, for control purposes. Myocardial infarction was confirmed by troponinI concentrations 24h after surgery. Peripheral blood was withdrawn and fractional shortening and ejection fraction of the left ventricle were assessed before (day0) and 14days after MI or sham surgery (day14). After mononuclear cell isolation, primary and secondary functional colony-forming unit granulocyte-macrophage (CFU-GM) assays were performed in order to detect the kinetics of functional HPC colony counts and cell self-renewal ability in vitro. The CFU-GM counts and cell self-renewal ability remained unchanged (P>0.05) in both groups at day14, without interaction between groups. In the intervention group, higher day0 CFU-GM counts showed a relationship to lower fractional shortening on day14 (ρ=-0.82; P<0.01). Myocardial infarction did not negatively affect circulating HPC self-renewal ability, which suggests a constant regenerative potential in the post-acute phase. A relationship of cardiac contractile function 14days after MI with circulating CFU-GM counts on day0 might imply functional colony count as a predictive factor for outcome after infarction.

Biomarker and Pharmacologic Evaluation of the γ-Secretase Inhibitor PF-03084014 in Breast Cancer Models

We aimed to assess the biologic activity of PF-03084014 in breast xenograft models. The biomarkers for mechanism and patient stratification were also explored. The in vitro and in vivo properties of PF-03084014 were investigated. The mRNA expressions of 40 key Notch pathway genes at baseline or after treatment were analyzed to link with the antitumor efficacy of PF-03084014 in a panel of breast cancer xenograft models. In vitro, PF-03084014 exhibited activity against tumor cell migration, endothelial cell tube formation, and mammosphere formation. In vivo, we observed apoptosis, antiproliferation, reduced tumor cell self-renewal ability, impaired tumor vasculature, and decreased metastasis activity after the treatment of PF-03084014. PF-03084014 treatment displayed significant antitumor activity in 10 of the 18 breast xenograft models. However, the antitumor efficacy in most models did not correlate with the in vitro antiproliferation results in the corresponding cell lines, suggesting the critical involvement of tumor microenvironment during Notch activation. In the tested breast xenograft models, the baseline expressions of the Notch receptors, ligands, and the cleaved Notch1 failed to predict the antitumor response to PF-03084014, whereas several Notch pathway target genes, including HEY2, HES4, and HES3, strongly corresponded with the response with a P value less than 0.01. Many of the best molecular predictors of response were also significantly modulated following PF-03084014 treatment. PF-03084014 showed antitumor and antimetastatic properties via pleiotropic mechanisms. The Notch pathway downstream genes may be used to predict the antitumor activity of PF-03084014 and enrich for responders among breast cancer patients.

Genetic Background Affects Properties of Satellite Cells and mdx Phenotypes

Duchenne muscular dystrophy (DMD) is the most common lethal genetic disorder of children. The mdx (C57BL/10 background, C57BL/10-mdx) mouse is a widely used model of DMD, but the histopathological hallmarks of DMD, such as the smaller number of myofibers, accumulation of fat and fibrosis, and insufficient regeneration of myofibers, are not observed in adult C57BL/10-mdx except for in the diaphragm. In this study, we showed that DBA/2 mice exhibited decreased muscle weight, as well as lower myofiber numbers after repeated degeneration-regeneration cycles. Furthermore, the self-renewal efficiency of satellite cells of DBA/2 is lower than that of C57BL/6. Therefore, we produced a DBA/2-mdx strain by crossing DBA/2 and C57BL/10-mdx. The hind limb muscles of DBA/2-mdx mice exhibited lower muscle weight, fewer myofibers, and increased fat and fibrosis, in comparison with C57BL/10-mdx. Moreover, remarkable muscle weakness was observed in DBA/2-mdx. These results indicate that the DBA/2-mdx mouse is a more suitable model for DMD studies, and the efficient satellite cell self-renewal ability of C57BL/10-mdx might explain the difference in pathologies between humans and mice.

BMI1 Interacts with FANCD2 at DNA Lesions and Prevents Chromosome Breaks

Bmi1 is a major determinant of hematopoietic stem cell (HSC) self-renewal. To date, much of our understanding of Bmi1 function comes from studies linking it to p16ink4a-Rb and p19arf-p53 pathways. However, several observations have raised the possibility that Bmi1 requirement for the maintenance of stem cell self-renewal ability extends far beyond its role in repressing the INK4a/ARF locus. The phenotypical similarities shared between Bmi1 deficiency in mouse and Fanconi Anemia (FA) patients (e.g., congenital aplastic anemia) together with the reported mono-ubiquitin ligase activities of proteins in both complexes and the emerging concept connecting DNA repair to stem cell properties, prompted us to evaluate Bmi1 implication in DNA damage repair. We now report that, like FA cells, Bmi1-deficient HSCs accumulate at G2/M phase of cell cycle. Moreover, Bmi1 is rapidly recruited to DNA damage foci in a PI3 kinase like kinase (PIKK)-dependant manner, in several cell types including hematopoietic stem cells. Upon induction of DNA lesions, Bmi1 interacts with several proteins involved in DNA damage response including γ-H2AX, MDC1, RAP80 and the monoubiquitinated form of FANCD2. We found that Bmi1 depletion did not hinder DNA damage-induced γ-H2AX foci formation and that RNF8-dependent signaling cascade is functional in absence of Bmi1. Furthermore, as observed in FA, loss of Bmi1 is associated with occurrence of spontaneous chromosome breaks and increased sensitivity to clastogenic agents. These chromosome breaks occur although FANCD2 monoubiquitination and loading onto chromatin is preserved. Together this work reveals an unsuspected role for Bmi1 in securing chromosome integrity and connects Polycomb genes to FA repair pathway.

Evidence of Both Ontogeny and Transplant Dose-Regulated Expansion of Hematopoietic Stem Cells In Vivo

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.