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(A) Morphologic and immunohistochemical characterization of NeoHep cells. Most NeoHep cells have a hexagonal shape (*), others appear to be in a transitional state resembling hepatoblasts or oval cells (3). (B) Expression of albumin in peroxidase-stained NeoHep cells. -1-antitrypsin was constantly expressed as demonstrated by alkaline phosphatase-anti-alkaline phosphatase (APAAP) stain in (C). A subpopulation of NeoHep cells, small oval cells (*), expressed CYP2D6 (D) and CYP2C9 (E). Immunofluorescence was carried out with fluorescein–isothiocyanate-coupled secondary antibody. Scale bars, 50 m.
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There is growing interest in new therapeutic options for the treatment of end-stage liver diseases. In addition to mechanical devices supporting liver function, such as bioreactors, the transplantation of hepatocyte-like cells derived from (adult) stem cells offer great perspectives. We have generated hepatocyte-like (NeoHep) cells from terminally...
Citations
... The stem cells injection induces liver tissue regeneration and repopulation by paracrine action of the injected cells (Lozito et al., 2009). Several growth factors and cytokines released during liver injury stimulate the migration of bone marrow cells (HSCs) to the injury site through circulation and hepatogenic differentiation and populate the liver after intravenous transplantation (Lagasse et al., 2000;Zhang et al., 2003;Ruhnke et al., 2005). Apart from that, EPCs from the bone marrow also reside at the sinusoidal endothelium (Kallis et al., 2007). ...
Extensive clinical efforts have been made to control the severity of dengue diseases;
however, the dengue morbidity and mortality have not declined. Dengue virus (DENV)
can infect and cause systemic damage in many organs, resulting in organ failure. Here,
we present a novel report showing a tailored stem-cell-based therapy that can aid in
viral clearance and rescue liver cells from further damage during dengue infection. We
administered a combination of hematopoietic stem cells and endothelial progenitor cells
in a DENV-infected BALB/c mouse model and found that delivery of this cell cocktail
had improved their liver functions, confirmed by hematology, histopathology, and next-
generation sequencing. These stem and progenitor cells can differentiate into target
cells and repair the damaged tissues. In addition, the regime can regulate endothelial
proliferation and permeability, modulate inflammatory reactions, enhance extracellular
matrix production and angiogenesis, and secrete an array of growth factors to create
an enhanced milieu for cell reparation. No previous study has been published on the
treatment of dengue infection using stem cells combination. In conclusion, dengue-
induced liver damage was rescued by administration of stem cell therapy, with less
apoptosis and improved repair and regeneration in the dengue mouse mod
... The stem cells injection induces liver tissue regeneration and repopulation by paracrine action of the injected cells (Lozito et al., 2009). Several growth factors and cytokines released during liver injury stimulate the migration of bone marrow cells (HSCs) to the injury site through circulation and hepatogenic differentiation and populate the liver after intravenous transplantation (Lagasse et al., 2000;Zhang et al., 2003;Ruhnke et al., 2005). Apart from that, EPCs from the bone marrow also reside at the sinusoidal endothelium (Kallis et al., 2007). ...
Extensive clinical efforts have been made to control the severity of dengue diseases; however, the dengue morbidity and mortality have not declined. Dengue virus (DENV) can infect and cause systemic damage in many organs, resulting in organ failure. Here, we present a novel report showing a tailored stem-cell-based therapy that can aid in viral clearance and rescue liver cells from further damage during dengue infection. We administered a combination of hematopoietic stem cells and endothelial progenitor cells in a DENV-infected BALB/c mouse model and found that delivery of this cell cocktail had improved their liver functions, confirmed by hematology, histopathology, and next-generation sequencing. These stem and progenitor cells can differentiate into target cells and repair the damaged tissues. In addition, the regime can regulate endothelial proliferation and permeability, modulate inflammatory reactions, enhance extracellular matrix production and angiogenesis, and secrete an array of growth factors to create an enhanced milieu for cell reparation. No previous study has been published on the treatment of dengue infection using stem cells combination. In conclusion, dengue-induced liver damage was rescued by administration of stem cell therapy, with less apoptosis and improved repair and regeneration in the dengue mouse model.
... Several studies have shown that hepatocyte-like cells can be generated from peripheral blood mononuclear cells (PBMCs) [1][2][3][4]. The procedure described by Ruhnke and colleagues initially involved a dedifferentiation step of the PBMCs in vitro to produce a cell type with stem cell-like characteristics termed "reprogrammed multipotent cells of monocytic origin" (RMCMO, formerly PCMO). ...
... The procedure described by Ruhnke and colleagues initially involved a dedifferentiation step of the PBMCs in vitro to produce a cell type with stem cell-like characteristics termed "reprogrammed multipotent cells of monocytic origin" (RMCMO, formerly PCMO). This step was followed by redifferentiation of RMCMO to either hepatocyte-like cells (neohepatocytes [3,4]) or insulin-producing cells [3] with appropriate differentiation media. ...
Following a several-day incubation in medium containing IL-3 and M-CSF to generate a more plastic intermediate "reprogrammed multipotent cells of monocytic origin (RMCMO)," peripheral blood mononuclear cells (PBMCs) can be efficiently converted to hepatocyte-like cells (neohepatocytes) and insulin-producing cells. However, continuous efforts are devoted to enhancing the proliferative capacity of these multipotent cells while maintaining or further increasing their redifferentiation potential. In the present work, PBMCs were transfected with one pluripotency gene (SOX2) and the resulting RMCMO compared to standard RMCMO with respect to cell viability, proliferative activity, and redifferentiation potential. Ectopic SOX2 expression increased the number of viable RMCMO, activated cell cycle genes, and enhanced proliferation as shown by quantitative RT-PCR and Ki67 immunofluorescent staining, respectively. Redifferentiation of RMCMO derived from SOX2-transfected PBMCs to neohepatocytes was more complete in comparison to control cells as revealed by higher urea and glucose secretion, increased activity of cytochrome P450 isoforms, and a phase II enzyme, while the same was true for insulin-producing cells as assessed by the expression of INS, PDX1, and GLUT2 and glucose-stimulated insulin secretion. Our results indicate that SOX2 transfection increases both multipotency and proliferation of RMCMO, eventually allowing production of neohepatocytes and insulin-producing cells of higher quality and quantity for transplantation purposes.
... Liver damage in chronic hepatitis C (CHC) is commonly attributed to immune-mediated mechanisms (7). Hepatic fibrosis is characterized by abnormal excessive accumulation of extracellular matrix accompanied by exaggerated cytokine release, However recruitment and trans-differentiation of peripheral blood cells, in particular, monocytes into injured liver may play a role in this respect (8). Chronic viral hepatitis is histologically characterized by intralobular infiltration of inflammatory cells which is considered as an ominous sign of deterioration and a criterion for disease activity (9). ...
... Moreover, HSCs persist in liver tissue throughout adulthood [85]. HSCs were shown to undergo hepatogenic differentiation and to populate liver after intravenous transplantation [34,86,87]. Similar data were reported by research groups working with MSC, another major bone marrow stem cell type [34,88,89] ( Table 1). ...
... First, blood-borne bone marrow-derived HSCs allegedly participate in liver regeneration [8,86,129]. Second, in animal models of liver malfunction infusion of HSC improves the outcomes [34,86,87]. Therapy with macrophages differentiating from HSCs improves outcomes of liver fibrosis in mice [130]. ...
The emerging field of regenerative medicine offers innovative methods of cell therapy and tissue/organ engineering as a novel approach to liver disease treatment. The ultimate scientific foundation of both cell therapy of liver diseases and liver tissue and organ engineering is delivered by the in-depth studies of the cellular and molecular mechanisms of liver regeneration. The cellular mechanisms of the homeostatic and injury-induced liver regeneration are unique. Restoration of the mass of liver parenchyma is achieved by compensatory hypertrophy and hyperplasia of the differentiated parenchymal cells, hepatocytes, while expansion and differentiation of the resident stem/progenitor cells play a minor or negligible role. Participation of blood-borne cells of the bone marrow origin in liver parenchyma regeneration has been proven but does not exceed 1-2% of newly formed hepatocytes. Liver regeneration is activated spontaneously after injury and can be further stimulated by cell therapy with hepatocytes, hematopoietic stem cells, or mesenchymal stem cells. Further studies aimed at improving the outcomes of cell therapy of liver diseases are underway. In case of liver failure, transplantation of engineered liver can become the best option in the foreseeable future. Engineering of a transplantable liver or its major part is an enormous challenge, but rapid progress in induced pluripotency, tissue engineering, and bioprinting research shows that it may be doable.
... e l s e v i e r . c o m / l o c a t e / t o x i n v i t SULT isoforms involved in sulfation of these substrates, however, are not characterized while glucuronidation is mainly catalyzed by UGT1A6 (Afolaranmi et al., 2011;Hengstler et al., 2000;Hewitt et al., 2000;Ruhnke et al., 2005;Tolando et al., 2012;Uchaipichat et al., 2004), thus ignoring the relative contribution of different relevant isoforms of these phase II enzymes. Cellular metabolism and toxicity studies using primary human hepatocytes can be performed using either suspension or monolayer incubation. ...
Cytochrome P450s (CYPS), UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) are the most important enzymes for metabolic clearance. Characterization of phase I and phase II metabolism of a given drug in cellular models is therefore important for an adequate interpretation of the role of drug metabolism in toxicity. We investigated phase I (CYP) and phase II (UGT and SULT) metabolism of three drugs related to drug-induced liver injury (DILI), namely acetaminophen (APAP), diclofenac (DF) and tolcapone (TC), in cryopreserved primary human hepatocytes from 5 donors in suspension and monolayer. The general phase II substrate 7-hydroxycoumarin (7-HC) was included for comparison. Our results show that the decrease in CYP, UGT and SULT activity after plating is substrate dependent. As a consequence the phase I/phase II metabolism ratio is significantly affected, with a shift in monolayer towards phase I metabolism for TC and towards phase II metabolism for APAP and DC. Inter-donor variability in drug metabolism is significant, especially in sulfation of 7-HC or APAP. As CYP, UGT and SULT metabolism may lead to bioactivation and/or detoxification of drugs, a changed ratio in phase I/phase II metabolism may have important consequences for metabolism-related toxicity.
... The functional plasticity and regenerative potential of monocytes/macrophages may be much greater than what is previously thought. Several cultured human cell populations that originate from circulating monocytes have the capacity to differentiate into nonphagocytic pluripotent stem cell-like cells such as pluripotent stem cells (PSCs) [2], MOMC [3,5], CD14+CD34 low KDR+ subset [10], and PCMO [4,11,12]. ...
... We have also shown earlier that PCMO generated from human peripheral blood or from either blood or spleen of a nonhuman primate can be converted to insulin-producing cells [4]. However, our focus was on PCMO-derived hepatocyte-like cells (so-called NeoHepatocytes), which express various hepatocyte markers and exhibit hepatocyte-specific metabolic functions in vitro and in vivo [4,11]. Intriguingly, NeoHepatocytes were able to improve survival in a rat model of acute liver failure [14] and monocyte-derived hepatocyte-like cells even showed promise in the treatment of HBV-related decompensated liver cirrhosis [15]. ...
Adult stem or programmable cells hold great promise in diseases in which damaged or nonfunctional cells need to be replaced. We have recently demonstrated that peripheral blood monocytes can be differentiated
in vitro
into cells resembling specialized cell types like hepatocytes and pancreatic beta cells. During phenotypic conversion, the monocytes downregulate monocyte/macrophage differentiation markers, being indicative of partial dedifferentiation, and are partially reprogrammed to acquire a state of plasticity along with expression of various markers of pluripotency and resumption of mitosis. Upregulation of stem cell markers and mitotic activity in the cultures was shown to be controlled by autocrine production/secretion of activin A and transforming growth factor-beta (TGF-
β
). These reprogrammed monocyte derivatives were termed “programmable cells of monocytic origin” (PCMO). Current efforts focus on establishing culture conditions that increase both the plasticity and proliferation potential of PCMO in order to be able to generate large amounts of blood-derived cells suitable for both autologous and allogeneic therapies.
... These cells have been suspected to be less mature and hence more stem cell-like than other monocytes [4]. PCMO are prone to acquire functional activities of hepatocyte-like cells (NeoHeps) and insulin-producing cells upon stimulation with appropriate differentiation media in vitro and in vivo following transplantation into mice [3,5]. ...
... Human peripheral blood monocytes were retrieved from buffy coats of healthy blood donors and cultured on tissue culture plastic as described in detail earlier [3][4][5][6][7]. Briefly, mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Amersham Pharmacia Biotech AB, Uppsala, Sweden) and cultured in 6-well plates (Cell+, Sarstedt, Numbrecht, Germany) for up to 4 days in RPMI 1640 medium (Life Technologies, Karlsruhe, Germany), supplemented with 5 ng/ml of M-CSF and 0.4 ng/ml of IL-3 (both from R&D Systems), 90 μM 2mercaptoethanol, and 10% human AB serum (Lonza, Verbier, Belgium). ...
... These cells have been suspected to be less mature and hence more stem cell-like than other monocytes [4]. PCMO are prone to acquire functional activities of hepatocyte-like cells (NeoHeps) and insulin-producing cells upon stimulation with appropriate differentiation media in vitro and in vivo following transplantation into mice [3,5]. ...
... Human peripheral blood monocytes were retrieved from buffy coats of healthy blood donors and cultured on tissue culture plastic as described in detail earlier [3][4][5][6][7]. Briefly, mononuclear cells were isolated by density gradient centrifugation (Ficoll-Paque; Amersham Pharmacia Biotech AB, Uppsala, Sweden) and cultured in 6-well plates (Cell+, Sarstedt, Numbrecht, Germany) for up to 4 days in RPMI 1640 medium (Life Technologies, Karlsruhe, Germany), supplemented with 5 ng/ml of M-CSF and 0.4 ng/ml of IL-3 (both from R&D Systems), 90 μM 2mercaptoethanol, and 10% human AB serum (Lonza, Verbier, Belgium). ...
Previous studies have shown that peripheral blood monocytes can be converted in vitro to a stem cell-like cell termed PCMO as evidenced by the re-expression of pluripotency-associated genes, transient proliferation, and the ability to adopt the phenotype of hepatocytes and insulin-producing cells upon tissue-specific differentiation. However, the regulatory interactions between cultured cells governing pluripotency and mitotic activity have remained elusive. Here we asked whether activin(s) and TGF-β(s), are involved in PCMO generation. De novo proliferation of PCMO was higher under adherent vs. suspended culture conditions as revealed by the appearance of a subset of Ki67-positive monocytes and correlated with down-regulation of p21WAF1 beyond day 2 of culture. Realtime-PCR analysis showed that PCMO express ActRIIA, ALK4, TβRII, ALK5 as well as TGF-β1 and the βA subunit of activin. Interestingly, expression of ActRIIA and ALK4, and activin A levels in the culture supernatants increased until day 4 of culture, while levels of total and active TGF-β1 strongly declined. PCMO responded to both growth factors in an autocrine fashion with intracellular signaling as evidenced by a rise in the levels of phospho-Smad2 and a drop in those of phospho-Smad3. Stimulation of PCMO with recombinant activins (A, B, AB) and TGF-β1 induced phosphorylation of Smad2 but not Smad3. Inhibition of autocrine activin signaling by either SB431542 or follistatin reduced both Smad2 activation and Oct4A/Nanog upregulation. Inhibition of autocrine TGF-β signaling by either SB431542 or anti-TGF-β antibody reduced Smad3 activation and strongly increased the number of Ki67-positive cells. Furthermore, anti-TGF-β antibody moderately enhanced Oct4A/Nanog expression. Our data show that during PCMO generation pluripotency marker expression is controlled positively by activin/Smad2 and negatively by TGF-β/Smad3 signaling, while relief from growth inhibition is primarily the result of reduced TGF-β/Smad3, and to a lesser extent, activin/Smad2 signaling.
... Stem cells and alternative cell sources for liver therapy In addition to attempts to improve engraftment and repopulation of the liver, alternative cell sources for hepatocytes have been proposed. Xenotransplants ( Nagata et al., 2003a), immortalized human hepatocytes ( Kobayashi et al., 2000;Cai et al., 2002;Wege et al., 2003a,b), stem cell-derived hepatocytes ( Avital et al., 2002;Miki et al., 2002Miki et al., , 2005Davila et al., 2004;Ruhnke et al., 2005), and fetal hepatocytes have been proposed as alternative sources of cells for clinical transplants. To date, no alternative cell source has been found that meets all of the requirements for safety and efficacy. ...
Authentic hepatocytes are the preferred cell type for the treatment of liver disease. Even 2–4-fold increases in liver repopulation by hepatocytes over the levels obtained with current transplant procedures could lead to substantial improvement in the clinical outcome of patients with liver-based metabolic diseases. It is likely that the incorporation of preconditioning regimens with hepatic resection, ischemia/reperfusion injury, and/or radiation-induced blockage of the growth of native liver provides the selective growth advantage to the donor cells required to attain the levels of liver repopulation required to normalize the alterations observed in metabolic disease patients. Hepatocyte transplantation studies conducted in animal models of liver failure and liver-based metabolic disease have proven safe and effective means to provide short- or long-term synthetic and metabolic support of liver function. For certain organ transplant candidates such as those with metabolic liver disease, cell transplantation alone could provide relief of the clinical symptoms. Cell transplant studies in patients with acute or chronic liver failure or genetic defects in liver function clearly demonstrate the efficacy of hepatocyte transplantation to treat liver disease. In virtually all cases, a clinical improvement in the condition of the patient could be documented. Studies in animal models of liver disease have documented that donor hepatocytes transplanted into the spleen or the liver function for the lifetime of the recipient and participate in normal regenerative events.
