Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

Hyperprogression under treatment with immune-checkpoint inhibitors in patients with gastrointestinal cancer: A natural process of advanced tumor progression?

Immunotherapy has shown great promise in treating various types of malignant tumors. However, some patients with gastrointestinal cancer have been known to experience rapid disease progression after treatment, a situation referred to as hyperprogressive disease (HPD). This minireview focuses on the definitions and potential mechanisms of HPD, natural disease progression in gastrointestinal malignancies, and tumor immunological microenvironment.

Targeted up-regulation of Drp1 in dorsal horn attenuates neuropathic pain hypersensitivity by increasing mitochondrial fission.

Mitochondria play an essential role in pathophysiology of both inflammatory and neuropathic pain (NP), but the mechanisms are not yet clear. Dynamin-related protein 1 (Drp1) is broadly expressed in the central nervous system and plays a role in the induction of mitochondrial fission process. Spared nerve injury (SNI), due to the dysfunction of the neurons within the spinal dorsal horn (SDH), is the most common NP model. We explored the neuroprotective role of Drp1 within SDH in SNI. SNI mice showed pain behavior and anxiety-like behavior, which was associated with elevation of Drp1, as well as increased density of mitochondria in SDH. Ultrastructural analysis showed SNI induced damaged mitochondria into smaller perimeter and area, tending to be circular. Characteristics of vacuole in the mitochondria further showed SNI induced the increased number of vacuole, widened vac-perimeter and vac-area. Stable overexpression of Drp1 via AAV under the control of the Drp1 promoter by intraspinal injection (Drp1 OE) attenuated abnormal gait and alleviated pain hypersensitivity of SNI mice. Mitochondrial ultrastructure analysis showed that the increased density of mitochondria induced by SNI was recovered by Drp1 OE which, however, did not change mitochondrial morphology and vacuole parameters within SDH. Contrary to Drp1 OE, down-regulation of Drp1 in the SDH by AAV-Drp1 shRNA (Drp1 RNAi) did not alter painful behavior induced by SNI. Ultrastructural analysis showed the treatment by combination of SNI and Drp1 RNAi (SNI + Drp1 RNAi) amplified the damages of mitochondria with the decreased distribution density, increased perimeter and area, as well as larger circularity tending to be more circular. Vacuole data showed SNI + Drp1 RNAi increased vacuole density, perimeter and area within the SDH mitochondria. Our results illustrate that mitochondria within the SDH are sensitive to NP, and targeted mitochondrial Drp1 overexpression attenuates pain hypersensitivity. Drp1 offers a novel therapeutic target for pain treatment.

Decreased spinal endomorphin-2 contributes to mechanical allodynia in streptozotocin-induced diabetic rats.

Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of diabetes patients, however, the neural mechanisms underlying DNP remain unclear. Endomorphins are the endogenous ligands for mu-opioid receptor. There is increasing evidence implicating the involvement of spinal endomorphin-2 (EM2) in neuropathic pain. In this study, using a streptozotocin induced diabetic rat model that displayed obvious mechanical allodynia, it was found that the expression of spinal EM2 was significantly decreased in DNP rats. While intrathecal administration of exogenous EM2 attenuated mechanical allodynia in DNP rats, the mu-opioid receptor antagonist ß-funaltrexamine facilitated these events. It was found that the reduction in spinal EM2 was mediated by increased activity of dipeptidylpeptidase IV, possibly as a consequence of diabetes-induced oxidative stress. Taken together, our results provide the first evidence that the reduction in the level of an endogenous opioid in primary afferents was significantly associated with DNP. This indicates that the chronic pain associated with DNP might be due to the loss of an inhibitory effect on pain signal transmission.

Antinociceptive effects of endomorphin-2: suppression of substance P release in the inflammatory pain model rat.

Endomorphin-2 (EM2) and Substance P (SP) exert suppressive and facilitative influences upon nociception, respectively. Although EM2 and SP were often co-expressed in single neurons in dorsal root ganglion (DRG), it is still unknown if and how the nociception-suppressive influences of EM2 might be exerted upon nociception-facilitative effects of SP in the DRG neurons. We examined these issues in the inflammatory pain model rats produced by subcutaneous injection of the complete Freund's adjuvant into the hind paw. The paw withdrawal threshold for mechanical allodynia was measured. Changes of EM2 and SP release were estimated by measuring intrathecal levels of EM2 and SP through in vivo microdialysis analysis of cerebrospinal fluid. The mechanical allodynia was dose-dependently attenuated by intrathecal injection of EM2 or a neurokinin-1 receptor antagonist, and facilitated by intrathecal injection of SP or a mu-opioid receptor (MOR) antagonist. Importantly, intrathecal level of SP was found to be lowered by intrathecal injection of EM2. Morphologically, colocalization of EM2-, MOR- and SP-immunoreactivity in single DRG neurons was observed by immunofluorescent histochemistry, and co-expression of EM2 and SP in large, dense-cored presynaptic vesicles in primary afferents, as well as localization of MOR on pre- and postsynaptic membrane in spinal dorsal horn, was also confirmed electron miscroscopically. Thus, the results indicated that analgesic influences of EM2 upon inflammatory pain might be exerted through suppression of SP release, supporting the assumptions that binding of EM2 to presynaptic MOR might induce such effects.

Increases in PKC gamma expression in trigeminal spinal nucleus is associated with orofacial thermal hyperalgesia in streptozotocin-induced diabetic mice.

Painful diabetic polyneuropathy (PDN) at the early phrase of diabetes frequently exhibits increased responsiveness to nociception. In diabetic patients and animal models, alterations in the transmission of orofacial sensory information have been demonstrated in trigeminal system. Herein, we examined the changes of protein kinase Cγ subunit (PKCγ) in trigeminal spinal nucleus (Sp5C) and observed the development of orofacial thermal sensitivity in streptozotocin (STZ)-induced type 1 diabetic mice. With hyperglycemia and body weight loss, STZ mice exhibited orofacial thermal hyperalgesia, along with increased PKCγ expression in Sp5C. Insulin treatment at the early stage of diabetes could alleviate the orofacial thermal hyperalgesia and impaired increased PKCγ in Sp5C in diabetic mice. In summary, our results demonstrate that PKCγ might be involved in orofacial thermal hyperalgesia of diabetes, and early insulin treatment might be effective way to treat orofacial PDN.

Upregulated microRNA-92b regulates the differentiation and proliferation of EpCAM-positive fetal liver cells by targeting C/EBPß.

microRNAs (miRNAs) are short noncoding RNAs that negatively regulate gene expression. Although recent evidences have been indicated that their aberrant expression may play an important role in cancer stem cells, the mechanism of their deregulation in neoplastic transformation of liver cancer stem cells (LCSCs) has not been explored. In our study, the HCC model was established in F344 rats by DEN induction. The EpCAM(+) cells were sorted out from unfractionated fetal liver cells and liver cancer cells using the FACS analysis and miRNA expression profiles of two groups were screened through microarray platform. Gain-of-function studies were performed in vitro and in vivo to determine the role of miR-92b on proliferation and differentiation of the hepatic progenitors. In addition, luciferase reporter system and gene function analysis were used to predict miR-92b target. we found that miR-92b was highly downregulated in EpCAM(+) fetal liver cells in expression profiling studies. RT-PCR analysis demonstrated reverse correlation between miR-92b expression and differentiation degree in human HCC samples. Overexpression of miR-92b in EpCAM(+) fetal liver cells significantly increased proliferation and inhibited differentiation as well as in vitro and in vivo studies. Moreover, we verified that C/EBPß is a direct target of miR-92b and contributes to its effects on proliferation and differentiation. We conclude that aberrant expression of miR-92b can result in proliferation increase and differentiation arrest of hepatic progenitors by targeting C/EBPß.

Comprehensive application of modern technologies in precise liver resection.

BACKGROUND: Liver surgery has gone through the phases of wedge liver resection, regular resection of hepatic lobes, irregular and local resection, extracorporeal hepatectomy, hemi-extracorporeal hepatectomy and Da Vinci surgical system-assisted hepatectomy. Taking advantage of modern technologies, liver surgery is stepping into an age of precise liver resection. This review aimed to analyze the comprehensive application of modern technologies in precise liver resection. DATA SOURCE: PubMed search was carried out for English-language articles relevant to precise liver resection, liver anatomy, hepatic blood inflow blockage, parenchyma transection, and down-staging treatment. RESULTS: The 3D image system can imitate the liver operation procedures, conduct risk assessment, help to identify the operation feasibility and confirm the operation scheme. In addition, some techniques including puncture and injection of methylene blue into the target Glisson sheath help to precisely determine the resection. Alternative methods such as Pringle maneuver are helpful for hepatic blood inflow blockage in precise liver resection. Moreover, the use of exquisite equipment for liver parenchyma transection, such as cavitron ultrasonic surgical aspirator, ultrasonic scalpel, Ligasure and Tissue Link is also helpful to reduce hemorrhage in liver resection, or even operate exsanguinous liver resection without blocking hepatic blood flow. Furthermore, various down-staging therapies including transcatheter arterial chemoembolization and radio-frequency ablation were appropriate for unresectable cancer, which reverse the advanced tumor back to early phase by local or systemic treatment so that hepatectomy or liver transplantation is possible. CONCLUSIONS: Modern technologies mentioned in this paper are the key tool for achieving precise liver resection and can effectively lead to maximum preservation of anatomical structural integrity and functions of the remnant liver. In addition, large randomized trials are needed to evaluate the usefulness of these technologies in patients with hepatocellular carcinoma who have undergone precise liver resection.

Spinal astrocytic activation contributes to mechanical allodynia in a rat chemotherapy-induced neuropathic pain model.

Chemotherapy-induced neuropathic pain (CNP) is the major dose-limiting factor in cancer chemotherapy. However, the neural mechanisms underlying CNP remain enigmatic. Accumulating evidence implicates the involvement of spinal glia in some neuropathic pain models. In this study, using a vincristine-evoked CNP rat model with obvious mechanical allodynia, we found that spinal astrocyte rather than microglia was dramatically activated. The mechanical allodynia was dose-dependently attenuated by intrathecal administratration of L-α-aminoadipate (astrocytic specific inhibitor); whereas minocycline (microglial specific inhibitor) had no such effect, indicating that spinal astrocytic activation contributes to allodynia in CNP rat. Furthermore, oxidative stress mediated the development of spinal astrocytic activation, and activated astrocytes dramatically increased interleukin-1ß expression which induced N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal neurons to strengthen pain transmission. Taken together, our findings suggest that spinal activated astrocytes may be a crucial component of the pathophysiology of CNP and "Astrocyte-Cytokine-NMDAR-neuron" pathway may be one detailed neural mechanisms underlying CNP. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for treating CNP.

Beneficial effects of splenectomy on liver regeneration in a rat model of massive hepatectomy.

BACKGROUND: Small-for-size syndrome is a widely recognized clinical complication after living donor liver transplantation or extended hepatectomy due to inadequate liver mass. The purpose of this study was to investigate the role of splenectomy in rats after massive hepatectomy, a surrogate model of small-for-size graft. METHODS: Rats were divided into eight groups, each with 20 animals: 50% hepatectomy (50% Hx), 50% hepatectomy+splenectomy (50% Hx+Sp), 60% Hx, 60% Hx+Sp, 70% Hx, 70% Hx+Sp, 90% Hx and 90% Hx+Sp. The following parameters were evaluated: liver function tests (ALT, AST and TBIL), liver regeneration ratio, DNA synthesis, proliferation cell nuclear antigen, hepatic oxygen delivery (HDO2) and hepatic oxygen consumption (HVO2). RESULTS: The liver regeneration ratio was enhanced in the Hx+Sp groups (P<0.05). In addition, compared with the Hx groups, the Hx+Sp groups had better liver functions (P<0.05). DNA synthesis and proliferation cell nuclear antigen were also increased in the Hx+Sp groups compared with the Hx groups (P<0.05). Furthermore, in the Hx+Sp groups, HDO2 and HVO2 were increased over those in the Hx groups (P<0.05), and were positively correlated with the liver regeneration ratio. CONCLUSIONS: Splenectomy significantly improved liver function, and enhanced DNA synthesis and proliferation cell nuclear antigen after massive hepatectomy in rats. This operation could be mediated through increased HDO2 and HVO2, which facilitate liver regeneration.

Sodium channel Nav1.6 is up-regulated in the dorsal root ganglia in a mouse model of type 2 diabetes.

Neuropathic pain is one of the most common chronic complications of diabetes, of which the underlying mechanisms are unclear. Expression changes of voltage-gated sodium channels in dorsal root ganglia (DRG) are involved in the production of ectopic spontaneous activity. In the present study, we examined the changes of DRG Nav1.6 expression in a mouse model of type 2 diabetes (db/db mice). Db/db mice developed significant and persistent mechanical allodynia from postnatal 2 months compared to the heterozygous littermates (db/+) and C57 mice. Immunofluorescent staining showed that Nav1.6 was highly expressed in the normal DRG (approximately 31.3±5.2% of total DRG neurons), especially in the large-diameter neurons. In postnatal 5 months in db/db mice, percentage of Nav1.6 positive cells (62.9±5.5%) was significantly higher than that in C57 and db/+ mice. Western blot showed that from 2 to 5 months, Nav1.6 was increased by 1.67±0.16, 2.12±0.23, 1.89±0.32, and 2.01±0.35 folds of C57 mice, which were significantly higher than that of the C57 and db/+ mice. Real-time PCR showed that in postnatal 1 month of db/db mice, mRNA level of Nav1.6 was increased by 1.72±0.22 fold, which was significantly higher than that of C57 and db/+ mice. Nav1.6 mRNA was increased thereafter and maintained at high levels throughout the observed period. Our results provide direct evidence that type 2 diabetes induces significant and persistent increase of Nav1.6 expression in the DRG, which may participate in the diabetic neuropathic pain.

Spinal astrocytic activation is involved in a virally-induced rat model of neuropathic pain.

Postherpetic neuralgia (PHN), the most common complication of herpes zoster (HZ), plays a major role in decreased life quality of HZ patients. However, the neural mechanisms underlying PHN remain unclear. Here, using a PHN rat model at 2 weeks after varicella zoster virus infection, we found that spinal astrocytes were dramatically activated. The mechanical allodynia and spinal central sensitization were significantly attenuated by intrathecally injected L-α-aminoadipate (astrocytic specific inhibitor) whereas minocycline (microglial specific inhibitor) had no effect, which indicated that spinal astrocyte but not microglia contributed to the chronic pain in PHN rat. Further study was taken to investigate the molecular mechanism of astrocyte-incudced allodynia in PHN rat at post-infection 2 weeks. Results showed that nitric oxide (NO) produced by inducible nitric oxide synthase mediated the development of spinal astrocytic activation, and activated astrocytes dramatically increased interleukin-1ß expression which induced N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to strengthen pain transmission. Taken together, these results suggest that spinal activated astrocytes may be one of the most important factors in the pathophysiology of PHN and "NO-Astrocyte-Cytokine-NMDAR-Neuron" pathway may be the detailed neural mechanisms underlying PHN. Thus, inhibiting spinal astrocytic activation may represent a novel therapeutic strategy for clinical management of PHN.

Spinal toll like receptor 3 is involved in chronic pancreatitis-induced mechanical allodynia of rat.

BACKGROUND: Mechanisms underlying pain in chronic pancreatitis (CP) are incompletely understood. Our previous data showed that astrocytes were actively involved. However, it was unclear how astrocytic activation was induced in CP conditions. In the present study, we hypothesized that toll-like receptors (TLRs) were involved in astrocytic activation and pain behavior in CP-induced pain. RESULTS: To test our hypothesis, we first investigated the changes of TLR2-4 in the rat CP model induced by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Western blot showed that after TNBS infusion, TLR3, but not TLR2 or TLR4, was increased gradually and maintained at a very high level for up to 5 w, which correlated with the changing course of mechanical allodynia. Double immunostaining suggested that TLR3 was highly expressed on astrocytes. Infusion with TLR3 antisense oligodeoxynucleotide (ASO) dose-dependently attenuated CP-induced allodynia. CP-induced astrocytic activation in the spinal cord was also significantly suppressed by TLR3 ASO. Furthermore, real-time PCR showed that IL-1ß, TNF-α, IL-6 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in spinal cord of pancreatic rats. In addition, TLR3 ASO significantly attenuated CP-induced up-regulation of IL-1ß and MCP-1. CONCLUSIONS: These results suggest a probable "TLR3-astrocytes-IL-1ß/MCP-1" pathway as a positive feedback loop in the spinal dorsal horn in CP conditions. TLR3-mediated neuroimmune interactions could be new targets for treating persistent pain in CP patients.

Spinal astrocytic activation contributes to mechanical allodynia in a mouse model of type 2 diabetes.

Diabetic neuropathic pain (DNP) plays a major role in decreased life quality of type 2 diabetes patients, however, the molecular mechanisms underlying DNP remain unclear. Emerging research implicates the participation of spinal glial cells in some neuropathic pain models. However, it remains unknown whether spinal glial cells are activated under type 2 diabetic conditions and whether they contribute to diabetes-induced neuropathic pain. In the present study, using a db/db type 2 diabetes mouse model that displayed obvious mechanical allodynia, we found that spinal astrocyte but not microglia was dramatically activated. The mechanical allodynia was significantly attenuated by intrathecally administrated l-α-aminoadipate (astrocytic specific inhibitor) whereas minocycline (microglial specific inhibitor) did not have any effect on mechanical allodynia, which indicated that spinal astrocytic activation contributed to allodynia in db/db mice. Further study aimed to identify the detailed mechanism of astrocyte-induced allodynia in db/db mice. Results showed that spinal activated astrocytes dramatically increased interleukin (IL)-1ß expression which may induce N-methyl-D-aspartic acid receptor (NMDAR) phosphorylation in spinal dorsal horn neurons to enhance pain transmission. Together, these results suggest that spinal activated astrocytes may be a crucial component of mechanical allodynia in type 2 diabetes and "Astrocyte-IL-1ß-NMDAR-Neuron" pathway may be the detailed mechanism of astrocyte-induced allodynia. Thus, inhibiting astrocytic activation in the spinal dorsal horn may represent a novel therapeutic strategy for treating DNP.

Percutaneous injection of hemostatic agents for active liver hemorrhage.

BACKGROUND: Active hemorrhage arising from hepatic injury can be life-threatening and require immediate attention. At present, nonoperative management of abdominal solid organ injuries has become the usual method of care. The purpose of this study was to determine whether hemocoagulase injection alone guided by contrast-enhanced ultrasonography (CEUS) could control active bleeding in rabbit liver. METHODS: The livers of 30 rabbits were punctured with an 18-gauge semiautomatic biopsy needle to create an active bleeding liver model, which was confirmed with CEUS. The animals were randomly divided into two groups: a treatment group (n=15) and a control group (n=15). In the treatment group, hemocoagulase was injected into the bleeding site under CEUS guidance. In the control group, the active bleeding site was treated with normal saline. When these treatment procedures had been performed, lactated Ringer's solution was given to both groups to maintain the mean arterial pressure at 70 mmHg for 1 hour. The intraperitoneal blood loss, hematocrit, mean heart rate, and macroscopic and microscopic examinations were analyzed at the end of the study. RESULTS: CEUS showed hypoechoic and anechoic perfusion defects in active bleeding liver models. Macroscopic and microscopic examinations also supported the results. After the hemocoagulase injection, the former bleeding site appeared on CEUS as an area devoid of contrast. The blood loss was lower in the treatment group than in the control group (38.0+/-16.6 ml versus 107.9+/-20.8 ml; t=10.172, P<0.05). The mean hematocrit value and the heart rate were higher in the treatment group than in the control group (hematocrit: 23.9+/-3.8% versus 18.8+/-4.1%; t=3.541, P<0.05; heart rate: 250+/-18 versus 223+/-15; t=4.551, P<0.01). CONCLUSION: Hemocoagulase injection alone under the guidance of CEUS is a simple and quick method to control blood loss in active liver bleeding.

Protective effect of cyclosporin A on brain injury in rats with acute necrotic pancreatitis.

AIMS: This study was designed to evaluate the protective effect of cyclosporin A (CsA) on brain injury in rats with acute necrotic pancreatitis (ANP) in order to provide a scientific basis for the use of the drug in treating brain injury caused by pancreatitis. MAIN METHODS: The rats were divided into a sham group, an ANP group and an ANP+CsA group. The ANP model was induced by administering 5% sodium taurocholate through the biliopancreatic duct. Five minutes before the preparation of the ANP model, 1 ml of CsA (10mg/kg) was injected in a clinically used pharmaceutical formulation (Sandimmun) via the dorsal vein of the penis. Twelve hours after the model was established, samples were taken from the rats in all groups for measurement of appropriate indexes. The serum levels of pro-inflammatory tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), and anti-inflammatory interleukin 10 (IL-10) were determined by ELISA. The pancreatic mRNA expressions of these cytokines were evaluated by RT-PCR. Brain water content was tested by the dry-wet method, and brain malondialdehyde (MDA) content was detected by the chemical colorimetry method. KEY FINDINGS: Both the serum levels and the pancreatic tissue mRNA expression of TNF-alpha and IL-1beta, as well as the brain water content and brain MDA content, were significantly increased in the ANP group. CsA treatment noticeably reduced both the serum levels and the pancreatic mRNA expression of TNF-alpha and IL-1beta and decreased brain water and MDA contents. In contrast to the pro-inflammatory cytokines, the serum levels and the pancreatic tissue mRNA expression of IL-10 were markedly increased by the injection of CsA. SIGNIFICANCE: CsA could alleviate acute pancreatitis-associated brain injury.

Replacing Hoechst33342 with rhodamine123 in isolation of cancer stem-like cells from the MHCC97 cell line.

The side population (SP) is commonly applied to analyzing cancer stem cells (CSCs) biology. However, the Hoechst33342 (Hoechst) dye, which is used for SP isolation, is demonstrated to be toxic to cells. The aim of this study was to identify a dye that might replace or offer an alternative to Hoechst use. In the trial group, by FACS binding Rhodamine123 (Rho), Rho(low) (low Rho fluorescence) and Rho(high) (high Rho fluorescence) were sorted from the MHCC97 cell line. In the control group, SP (low Hoechst fluorescence) and NSP (high Hoechst fluorescence) were obtained through Hoechst/FACS. The percentages of subpopulations were as following: Rho(low) 2.1%, Rho(high) 97.9%, SP 1.4%, NSP 98.6%. The proliferative abilities in vitro (growth rate, soft agar clone formation rate), expressions of stem cell markers (early hepatic marker AFP and CSCs marker CD133), and tumorigenicities in vivo were significantly higher in the Rho(low) and SP cells than those in the Rho(high) and NSP cells, respectively (P<0.05). In addition, each result was the same between the Rho(low) and SP cells (P>0.05). Taken together, Rho/FACS may not be as effective as Hoechst/FACS for CSCs sorting, but given its low toxicity and cost, it may be a useful method for CSCs identification.