Adult-Derived Human Liver Mesenchymal-Like Cells as a Potential Progenitor Reservoir of Hepatocytes?

It is currently accepted that adult tissues may develop and maintain their own stem cell pools. Because of their higher safety profile, adult stem cells may represent an ideal candidate cell source to be used for liver cell therapies. We therefore evaluated the differentiation potential of mesenchymal-like cells isolated from adult human livers. Mesenchymal-like cells were isolated from enzymatically digested adult human liver and expanded in vitro. Cell characterization was performed using flow cytometry, RT-PCR, and immunofluorescence, whereas the differentiation potential was evaluated both in vitro after incubation with specific media and in vivo after intrasplenic transplantation of uPA+/+-SCID and SCID mice. Adult-derived human liver mesenchymal-like cells expressed both hepatic and mesenchymal markers among which albumin, CYP3A4, vimentin, and α-smooth muscle actin. In vitro differentiation studies demonstrated that these mesenchymal-like cells are preferentially determined to differentiate into hepatocyte-like cells. Ten weeks following intrasplenic transplantation into uPA+/+-SCID mice, recipient livers showed the presence of human hepatocytic cell nodules positive for human albumin, prealbumin, and α-fetoprotein. In SCID transplanted liver mice, human hepatocyte-like cells were mostly found near vascular structures 56 days posttransplantation. In conclusion, the ability of isolated adult-derived liver mesenchymal stem-like cells to proliferate and differentiate into hepatocyte-like cells both in vitro and in vivo leads to propose them as an attractive expandable cell source for stem cell therapy in human liver diseases.

Download Full-text

Are MSCs Stem Cells? Demonstration of in Vitro and in Vivo Stem Cell Properties of Highly Enriched linneg/CD45neg/CD271pos/CD140alow/Neg Mesenchymal Stromal Cells from Adult Human Bone Marrow

Blood ◽

10.1182/blood.v124.21.4374.4374 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4374-4374

Author(s):

Roshanak Ghazanfari ◽

Hongzhe Li ◽

Dimitra Zacharaki ◽

Simón Méndez-Ferrer ◽

Stefan Scheding

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Human Bone ◽

Human Bone Marrow ◽

Differentiation Potential ◽

Self Renewal ◽

Adult Human

Abstract Human bone marrow contains a rare population of non-hematopoietic mesenchymal stromal cells (BM-MSC) with multilineage differentiation capacity, which are essential constituents of the hematopoietic microenvironment. Self-renewal and differentiation are the two key properties of somatic stem cells, however, stem cell properties of human adult BM-MSC have not been demonstrated conclusively yet. We have previously shown that low/negative expression of PDGFRα on linneg/CD45neg/CD271pos cells identified a highly enriched population of primary BM-MSC in adult human bone marrow (Li et al. Blood, 2013, 122:3699). Based on this work, the current study aimed to investigate the in-vitro and in-vivo stem cell properties of this putative stromal stem cell population. The in-vitro clonogenic potential of freshly sorted human linneg/CD45neg/CD271pos/PDGFRlow/neg cells was evaluated by utilizing the CFU-F assay as well as the recently-developed mesensphere assay, which enables MSC amplification while preserving an immature phenotype (Isern et al, Cell Reports 2013, 30: 1714-24). Comparable colony frequencies were obtained with both assays (19.3 ± 2 and 17.5 ± 2.3 CFU-F and spheres per 100 plated cells, respectively, n=6, p=0.19). In order to test whether both assays identified the same population of clonogenic cells, colonies and spheres were replated under both conditions for up to three generations. The results showed comparable capacities of CFU-F and mesenspheres to form secondary and tertiary CFU-F and spheres. In-vitro self-renewal as indicated by increasing numbers of CFU-F and spheres (416.6 ± 431.7-fold and 49.5 ± 65.7-fold, respectively, n=3) was observed up to the third generation and decreased thereafter. The total number of generations was five (CFU-F) and six (spheres). In-vitro differentiation assays with both, CFU-F- and sphere-derived cells (tested until passage three) demonstrated tri-lineage differentiation potential (adipocytes, osteoblasts, chondrocytes). In addition, CFU-Fs and spheres had comparable surface marker profiles (CD73, CD90, CD105, and HLA-ABC positive; CD31, CD34 and HLA-DR negative), except for CD90, which was higher expressed on CFU-Fs. To investigate in-vivo self-renewal and differentiation potential of the putative stromal stem cells, linneg/CD45neg/CD271pos/PDGFRlow/neg -derived CFU-F and spheres were serially transplanted s.c into NSG mice. After 8 weeks, implants were harvested, human cells were FACS-isolated (CD90 and CD105 expression), and re-assayed under CFU-F and sphere conditions. Whereas in-vivo self-renewal of CFU-F could not be shown (111.5 ± 36 –fold decrease in total CFU-F numbers after primary transplantation, n=3), sphere self-renewal was clearly demonstrated by increased numbers of spheres after primary as well as secondary transplantation (1.13 ± 0.05 and 2.06 ± 0.26 –fold, respectively, n=3), which is remarkable given the fact that the number of recovered human cells is underestimated due to the isolation approach. Here, confirming GFP-marking experiments are ongoing. Finally, preliminary data indicate that linneg/CD45neg/CD271pos/PDGFRlow/neg –derived spheres display full in-vivo differentiation capacity in primary and secondary transplantations. Taken together, our data demonstrate - for the first time - that primary human linneg/CD45neg/CD271pos/PDGFRlow/neg cells meet stringent stem cell criteria, i.e. in-vitro and in-vivo self-renewal and differentiation. These findings answer the long-open question of the potential stem cell properties of adult human MSC and will enable to better understand the properties of native BM-MSC and their biological role in the bone marrow. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Non-Ionizing Radiation for Cardiac Human Amniotic Mesenchymal Stromal Cell Commitment: A Physical Strategy in Regenerative Medicine

International Journal of Molecular Sciences ◽

10.3390/ijms19082324 ◽

2018 ◽

Vol 19 (8) ◽

pp. 2324 ◽

Cited By ~ 1

Author(s):

Mario Ledda ◽

Enrico D’Emilia ◽

Maria Lolli ◽

Rodolfo Marchese ◽

Claudio De Lazzari ◽

...

Keyword(s):

Stem Cell ◽

Cell Therapy ◽

Adult Stem Cells ◽

Myocardial Damage ◽

Stem Cell Differentiation ◽

Differentiation Markers ◽

Innovative Strategy ◽

Cell Commitment

Cell therapy is an innovative strategy for tissue repair, since adult stem cells could have limited regenerative ability as in the case of myocardial damage. This leads to a local contractile dysfunction due to scar formation. For these reasons, refining strategy approaches for “in vitro” stem cell commitment, preparatory to the “in vivo” stem cell differentiation, is imperative. In this work, we isolated and characterized at molecular and cellular level, human Amniotic Mesenchymal Stromal Cells (hAMSCs) and exposed them to a physical Extremely Low Frequency Electromagnetic Field (ELF-EMF) stimulus and to a chemical Nitric Oxide treatment. Physically exposed cells showed a decrease of cell proliferation and no change in metabolic activity, cell vitality and apoptotic rate. An increase in the mRNA expression of cardiac and angiogenic differentiation markers, confirmed at the translational level, was also highlighted in exposed cells. Our data, for the first time, provide evidence that physical ELF-EMF stimulus (7 Hz, 2.5 µT), similarly to the chemical treatment, is able to trigger hAMSC cardiac commitment. More importantly, we also observed that only the physical stimulus is able to induce both types of commitments contemporarily (cardiac and angiogenic), suggesting its potential use to obtain a better regenerative response in cell-therapy protocols.

Download Full-text

Human NK cell development in NOD/SCID mice receiving grafts of cord blood CD34+ cells

Blood ◽

10.1182/blood-2002-07-2024 ◽

2003 ◽

Vol 102 (1) ◽

pp. 127-135 ◽

Cited By ~ 30

Author(s):

Christian P. Kalberer ◽

Uwe Siegler ◽

Aleksandra Wodnar-Filipowicz

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Scid Mouse ◽

Nk Cell ◽

Cell Development ◽

Scid Mice ◽

Differentiation Potential ◽

Ifn Γ

Abstract Definition of the cytokine environment, which regulates the maturation of human natural killer (NK) cells, has been largely based on in vitro assays because of the lack of suitable animal models. Here we describe conditions leading to the development of human NK cells in NOD/SCID mice receiving grafts of hematopoietic CD34+ precursor cells from cord blood. After 1-week-long in vivo treatment with various combinations of interleukin (IL)–15, flt3 ligand, stem cell factor, IL-2, IL-12, and megakaryocyte growth and differentiation factor, CD56+CD3- cells were detected in bone marrow (BM), spleen, and peripheral blood (PB), comprising 5% to 15% of human CD45+ cells. Human NK cells of NOD/SCID mouse origin closely resembled NK cells from human PB with respect to phenotypic characteristics, interferon (IFN)–γ production, and cytotoxicity against HLA class 1–deficient K562 targets in vitro and antitumor activity against K562 erythroleukemia in vivo. In the absence of growth factor treatment, CD56+ cells were present only at background levels, but CD34+CD7+ and CD34-CD7+ lymphoid precursors with NK cell differentiation potential were detected in BM and spleen of chimeric NOD/SCID mice for up to 5 months after transplantation. Our results demonstrate that limitations in human NK cell development in the murine microenvironment can be overcome by treatment with NK cell growth–promoting human cytokines, resulting in the maturation of IFN-γ–producing cytotoxic NK cells. These studies establish conditions to explore human NK cell development and function in vivo in the NOD/SCID mouse model. (Blood. 2003;102:127-135)

Download Full-text

Osteogenic Potential of Multipotent Adult Progenitor Cells for Calvaria Bone Regeneration

Advances in Medicine ◽

10.1155/2016/2803081 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11

Author(s):

Dong Joon Lee ◽

Yonsil Park ◽

Wei-Shou Hu ◽

Ching-Chang Ko

Keyword(s):

Bone Regeneration ◽

Progenitor Cells ◽

Adult Stem Cells ◽

Single Cells ◽

Osteogenic Potential ◽

Type I ◽

Differentiation Potential ◽

Multipotent Adult Progenitor Cells

Osteogenic cells derived from rat multipotent adult progenitor cells (rMAPCs) were investigated for their potential use in bone regeneration. rMAPCs are adult stem cells derived from bone marrow that have a high proliferation capacity and the differentiation potential to multiple lineages. They may also offer immunomodulatory properties favorable for applications for regenerative medicine. rMAPCs were cultivated as single cells or as 3D aggregates in osteogenic media for up to 38 days, and their differentiation to bone lineage was then assessed by immunostaining of osteocalcin and collagen type I and by mineralization assays. The capability of rMAPCs in facilitating bone regeneration was evaluatedin vivoby the direct implantation of multipotent adult progenitor cell (MAPC) aggregates in rat calvarial defects. Bone regeneration was examined radiographically, histologically, and histomorphometrically. Results showed that rMAPCs successfully differentiated into osteogenic lineage by demonstrating mineralized extracellular matrix formationin vitroand induced new bone formation by the effect of rMAPC aggregatesin vivo. These outcomes confirm that rMAPCs have a good osteogenic potential and provide insights into rMAPCs as a novel adult stem cell source for bone regeneration.

Download Full-text

225 TERATOMA FORMATION BY BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab225 ◽

2007 ◽

Vol 19 (1) ◽

pp. 229

Author(s):

M. L. Lim ◽

I. Vassiliev ◽

P. J. Verma

Keyword(s):

Growth Factor ◽

Es Cells ◽

Calf Serum ◽

Embryonic Stem ◽

Scid Mice ◽

Bovine Embryos ◽

Differentiation Potential ◽

Teratoma Formation

Teratoma formation is commonly used as a model for examining the in vivo differentiation potential of embryonic stem cells. We wanted to investigate the teratoma-forming ability of bovine ES cells; however, there are no reports of teratoma-forming ability of bovine pluripotent cells including pre-implantation embryos. In vivo-produced bovine embryos at stages earlier than Day 14 failed to develop teratomas when transplanted into one of the kidneys of immuno-deficient mice (Anderson et al. 1996 Anim. Reprod. Sci. 45, 231–240), and this prompted questions about the ability of bovine embryos to form teratomas. Bovine oocytes were cultured for 20 to 22 h after aspiration at 39�C (5% CO2/95% air) in TCM-199-bicarbonate medium supplemented with GlutaMax6" (Invitrogen Australia Pty Ltd., Mount Waverley, Victoria, Australia), penicillin/streptomycin, β-mercaptoethanol, 17β-estradiol, fetal calf serum, LH, follicle stimulating hormone, basic fibroblast growth factor, epidermal growth factor, glycine, and l-cysteine. Oocytes were fertilized with IVF media (Cook Australia, Brisbane, Queensland, Australia) and kept for 7 days at 39�C in 5% CO2/95% air to generate blastocysts. The zona pellucida of Day 7 blastocysts was enzymatically removed, and one or two zona-free embryos were injected into each testis of 5-week-old immunodeficient (SCID) mice (CB-17/ICR-Prkdcscid strain; Walter and Eliza Hall Institute, Melbourne, Australia). Eight weeks post-injection, teratomas partially expelled from testes were identified. Histological analysis has confirmed the derivatives of all 3 germ layers in teratomas. In conclusion, we report that Day 7 in vitro-produced embryos can form teratomas when injected into testes of SCID mice.

Download Full-text

182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab182 ◽

2019 ◽

Vol 31 (1) ◽

pp. 215

Author(s):

M. Nowak-Imialek ◽

X. Gao ◽

P. Liu ◽

H. Niemann

Keyword(s):

Stem Cell ◽

Embryonic Stem Cell ◽

Cell Lines ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Differentiation Potential ◽

Germ Layers ◽

Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

Download Full-text

Ectopic Human Mesenchymal Stem Cell-Coated Scaffolds Create An In Vivo Leukemia Niche in NOD/SCID Mice.

Blood ◽

10.1182/blood.v114.22.559.559 ◽

2009 ◽

Vol 114 (22) ◽

pp. 559-559

Author(s):

Sarah Rivkah Vaiselbuh ◽

Morris Edelman ◽

Jeffrey Michael Lipton ◽

Johnson M. Liu

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Stem Cell Niche ◽

Scid Mice ◽

Cxcr4 Antagonist ◽

Cell Niche

Abstract Abstract 559 Introduction: Different cellular components of the normal hematopoietic niche have been identified. However, the niche for malignant hematopoiesis remains to be elucidated. Recent work of other groups has suggested that hematopoietic stem cells (HSC) within the bone marrow anchor themselves in place by attaching to osteoblasts and/or vascular sinusoid endothelial cells. We have recently identified mesenchymal stem cells (MSC) as niche-maker cells and found a crucial role of the SDF-1/CXCR4 axis in this process. Stromal Derived Factor-1 (SDF-1/CXCL12) regulates stem cell trafficking and the cell cycle via its receptor CXCR4. Methods: Polyurethane scaffolds, coated in vitro with human bone marrow MSC, were implanted subcutaneously in non-irradiated NOD/SCID mice. CD34+ HSC or primary AML cells (from a leukapheresis product) were injected either in situ or retro-orbitally in the mice and analyzed for engraftment. The mice were treated twice per week with in situ injections of SDF-1, AMD3100 (a CXCR4 antagonist) or PBS (control). After 2 to 4 weeks, the scaffolds were processed and evaluated for cell survival in the mesenchymal niche by immunohistochemistry. Results: We created in vitro MSC-coated scaffolds that retained inoculated AML cells in the presence of SDF-1, while AML cells seeded on empty scaffolds were not retained. In vivo in NOD/SCID mice, the MSC-coated scaffolds, in the presence of SDF-1 enabled homing of both in situ injected normal CD34+ HSC and retroorbital- or in situ injected primary human AML cells. The scaffolds were vascularized and showed osteoclasts and adipocytes present, suggestive of an ectopic human bone marrow microenvironment in the murine host. Finally, the SDF-1-treated scaffolds showed proliferation of the MSC stromal layer with multiple adherent AML cells, while in the AMD3100-treated scaffolds the stromal lining was thin and disrupted at several points, leaving AML cells free floating in proximity. The PBS-treated control-scaffold showed a thin single cell MSC stromal layer without disruption, with few AML cells attached. Conclusion: The preliminary data of this functional ectopic human microenvironment in NOD/SCID mice suggest that AMD3100 (a CXCR4 antagonist) can disrupt the stem cell niche by modulation of the mesenchymal stromal. Further studies are needed to define the role of mesenchymal stem cells in maintaining the hematopoietic/leukemic stem cell niche in vivo. In Vivo Leukemia Stem Cell Niche: (A) Empty polyurethane scaffold. (B)Vascularization in SQ implanted MSC-coated scaffold (s) niche in NOD/SCID mice. (C) DAB Peroxidase (brown) human CD45 positive nests of AML cells (arrows) 1 week after direct in situ AML injection. (D) Human CD45 positive myeloid cells adhere to MSC in vivo (arrows). Disclosures: No relevant conflicts of interest to declare.

Download Full-text

279 Human liver cancer stem cell resistance to sorafenib and enhanced sensitivity by MK2206 in vitro and in vivo

European Journal of Cancer ◽

10.1016/s0959-8049(16)30164-2 ◽

2015 ◽

Vol 51 ◽

pp. S52-S53

Author(s):

Z. Yin ◽

B. Zhai ◽

X. Zhang ◽

B. Sun ◽

L. Cao ◽

...

Keyword(s):

Stem Cell ◽

Liver Cancer ◽

Cancer Stem Cell ◽

Human Liver ◽

Cell Resistance ◽

Enhanced Sensitivity ◽

Human Liver Cancer ◽

Liver Cancer Stem Cell

Download Full-text

Chicken embryonic brain: an in vivo model for verifying neural stem cell potency

Journal of Neurosurgery ◽

10.3171/2013.1.jns12698 ◽

2013 ◽

Vol 119 (2) ◽

pp. 512-519 ◽

Cited By ~ 4

Author(s):

Alex Kharazi ◽

Michael L. Levy ◽

Maria Cristina Visperas ◽

Chih-Min Lin

Keyword(s):

Stem Cell ◽

Fetal Brain ◽

Stem Cell Marker ◽

In Vivo Model ◽

Embryonic Brain ◽

In Vitro Differentiation ◽

Differentiation Potential ◽

Marker Expression

Object The multipotency of neural stem cells (NSCs) can be assessed in vitro by detection of stage-specific markers in response to a suitable differentiation signal. This test is frequently used because it is fast and affordable. However, it is not clear how the in vitro potential for multilineage differentiation and stem cell marker expression would reflect the ability of NSCs to engraft into the brain following transplantation. The authors undertook this study to directly compare the in vitro potency and in vivo migration of human NSCs (hNSCs) expanded under conditions of gradually increased concentration of fetal bovine serum (FBS) as a maturation factor. Methods Human NSCs isolated from fetal brain were propagated in serum free media (SF-hNSCs) and in media containing 0.1% and 0.2% serum. At Passage 4 in tissue culture the NSCs were harvested and either differentiated in vitro or transplanted into the lateral ventricle of chicken embryonic brain at the late stage of its development (Hamburger and Hamilton Stage 26). The in vitro differentiation was evaluated by immunostaining with neural or glial specific markers, and the in vivo migration was assessed using immunohistology. Results The authors found that SF-hNSCs successfully engrafted into the chicken embryonic brain, which correlated with their ability to differentiate in vitro. NSCs grown at as low as 0.1% concentration of FBS failed to demonstrate the robust in vivo migration pattern but still preserved the capability to differentiate in vitro. Furthermore, NSCs generated in media containing a higher concentration of FBS (0.2%) lost both the in vivo engraftment and in vitro differentiation potential. Conclusions The present study suggests that marker expression and in vitro differentiation assays might not provide adequate information regarding the behavior of NSCs following their transplantation. The in vivo migration following injection into chicken embryonic brain may provide an important assay of the potency of NSCs.

Download Full-text