scholarly journals A Decellularized Human Limbal Scaffold for Limbal Stem Cell Niche Reconstruction

2021 ◽  
Vol 22 (18) ◽  
pp. 10067
Author(s):  
Naresh Polisetti ◽  
Benjamin Roschinski ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Günther Schlunck ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Host Tissue ◽  
Fibrin Gel ◽  
Deoxyribonuclease I ◽  
Limbal Stem Cell

The transplantation of ex vivo expanded limbal epithelial progenitor cells (LEPCs) on amniotic membrane or fibrin gel is an established therapeutic strategy to regenerate the damaged corneal surface in patients with limbal stem cell deficiency (LSCD), but the long-term success rate is restricted. A scaffold with niche-specific structure and extracellular matrix (ECM) composition might have the advantage to improve long-term clinical outcomes, in particular for patients with severe damage or complete loss of the limbal niche tissue structure. Therefore, we evaluated the decellularized human limbus (DHL) as a biomimetic scaffold for the transplantation of LEPCs. Corneoscleral tissue was decellularized by sodium deoxycholate and deoxyribonuclease I in the presence or absence of dextran. We evaluated the efficiency of decellularization and its effects on the ultrastructure and ECM composition of the human corneal limbus. The recellularization of these scaffolds was studied by plating cultured LEPCs and limbal melanocytes (LMs) or by allowing cells to migrate from the host tissue following a lamellar transplantation ex vivo. Our decellularization protocol rapidly and effectively removed cellular and nuclear material while preserving the native ECM composition. In vitro recellularization by LEPCs and LMs demonstrated the good biocompatibility of the DHL and intrastromal invasion of LEPCs. Ex vivo transplantation of DHL revealed complete epithelialization as well as melanocytic and stromal repopulation from the host tissue. Thus, the generated DHL scaffold could be a promising biological material as a carrier for the transplantation of LEPCs to treat LSCD.

scholarly journals A decellularized human corneal scaffold for anterior corneal surface reconstruction

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Naresh Polisetti ◽  
Anke Schmid ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Stefan J. Lang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Biological Properties ◽  
Host Cells ◽  
Human Cornea ◽  
Human Donor ◽  
Short Period

AbstractAllogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4641-4651 ◽  
Author(s):  
Pankaj Gupta ◽  
Theodore R. Oegema ◽  
Joseph J. Brazil ◽  
Arkadiusz Z. Dudek ◽  
Arne Slungaard ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Stem Cell Niche ◽  
Ex Vivo ◽  
Recent Observation ◽  
Hematopoietic Progenitors ◽  
Platelet Factor ◽  
Cell Niche

Abstract Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Pre-Transplant Integration Site Diagnostics in Hematopoietic Stem Cell Gene Transfer.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3581-3581
Author(s):  
Claudia R Ball ◽  
Sylvia Fessler ◽  
Daniela Belle ◽  
Manfred Schmidt ◽  
Christof von Kalle ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Integration Site ◽  
Hematopoietic Stem ◽  
Cell Clones ◽  
Integration Sites ◽  
Cell Gene ◽  
Stem Cell Gene

Abstract Abstract 3581 Poster Board III-518 We and others have previously shown that insertional activation of cellular genes caused by integrated retroviral vectors can lead to clonal dominance and malignant transformation. Pre-transplant diagnostics of vector flanking sequences and subsequent elimination of those clones that carry potentially dangerous integration sites prior to transplantation would dramatically improve the safety of clinical gene therapy regimens. Such a strategy requires efficient transduction of few or individual stem cells, their in vitro amplification and highly sensitive integration site determination before transplantation. To define optimal time points for transduction and ascertain the transplantability of ex vivo expanded murine stem cell clones, single CD45+Lin−Rho+SP cells isolated from bone marrow of male C57BL/6J (B6J) mice were cultivated for 8-10 days in the presence of IL11, SCF and Flt3-L. 10% of the sorted cells formed clones in vitro. In 28% ± 5% of these clones, the first division occurred during the first 48 hours after sorting, another 32% ± 8% divided up to 72 hours after sorting and additional 33% ± 7% up to 96 hours after sorting. 7% ± 4% had undergone their first division at a later time point. To examine the transplantability after ex vivo expansion, individual cell clones (containing 12 to >600 cells) were transplanted together with 105 carrier cells into lethally irradiated sex-mismatched syngeneic mice. The presence of donor-derived cells in peripheral blood of 20 transplanted mice was analyzed by Y-chromosome specific PCR. 55% of the ex vivo expanded clones contributed to post-transplant hematopoiesis. 25% of these clones exhibited long-term activity for >6 months after transplantation. Interestingly, only cell clones that had undergone their first division 48-96 hours after cell sorting contributed to long-term post-transplant hematopoiesis. For transduction, individual stem cell clones were spinoculated for 60 minutes with a GFP encoding lentiviral vector (MOI 100-5000). 5 days after transduction, 50% of cells generated by each clone were harvested, lysed and analyzed by LAM-PCR and integration site sequencing. After an additional 3 days, single clones were transplanted together with 105 carrier cells into lethally irradiated congeneic B6.SJL-PtprcaPepcb/BoyJ mice. Four weeks after transplantation, in 30% of these mice ≥0.4% CD45.1+ cells derived from single cell clones were detected in the peripheral blood. In 50% of these mice, the transduced clones contributed to myelopoiesis as well as lymphopoiesis for more than 24 weeks after transplantation, demonstrating that the longterm hematopoietic stem cell potential was retained after single cell marking and expansion. These results demonstrate that single stem cell gene transfer and subsequent expansion is possible to allow integration site determination. Long-term stem cells with defined lentiviral integration sites can be selected for transplantation. In summary, we provide proof of concept that pre-transplant diagnostics of integration sites is feasible to increase the safety of gene therapy by eliminating stem cell clones from transplants that carry unwanted integration sites. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A Decellularized Human Corneal Scaffold for Anterior Corneal Surface Reconstruction

2020 ◽  
Author(s):  
Naresh Polisetti ◽  
Anke Schmid ◽  
Ursula Schlötzer-Schrehardt ◽  
Philip Maier ◽  
Stefan Lang ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Ex Vivo ◽  
Sodium Deoxycholate ◽  
Nuclear Material ◽  
Biological Properties ◽  
Host Cells ◽  
Human Cornea ◽  
Human Donor ◽  
Short Period

Abstract Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

Reducing Stem Cell Loss and Ex Vivo Differentiation during Lentivirus Gene Transfer to Murine Stem Cells - an Ultra-Short Transduction Protocol.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2113-2113
Author(s):  
Peter Kurre ◽  
Ponni Anandakumar ◽  
Vladimir A. Lesnikov ◽  
Hans-Peter Kiem
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Lentivirus Vectors ◽  
Ex Vivo Culture ◽  
Marrow Cells

Abstract Most gene transfer models using Moloney murine leukemia virus (MLV) - derived vectors to target hematopoietic repopulating cells require progenitor cell enrichment and extended ex vivo culture for efficient long-term marking. Both may result in qualitative, and/or quantitative, loss of stem cells thereby limiting gene transfer rates in vivo. This can be a critical obstacle in candidate applications with exhausted autologous stem cell pools, such as Fanconi Anemia. Among the advantages of HIV-derived lentivirus vectors is their ability to transduce non dividing cells, permitting shortened ex vivo culture durations while maintaining gene transfer to long-term repopulating cells. We have previously reported long-term gene transfer rates of 12–40% after VSV-G/ lentivirus vector transduction of murine stem cells by targeting unseparated marrow cells after reduced prestimulation and a single 12 hour vector exposure (Kurre et al., Mol. Ther. 2004 Jun;9(6):914–22). We herein report studies showing maintenance of gene transfer efficiency in this model at drastically reduced ex vivo vector exposure times. In initial in vitro experiments we studied cytokine support, vector particle density, and minimum exposure duration requirements for efficient gene transfer to unseparated marrow cells. We determined that fibronectin fragment support was critical in maintaining minimum gene transfer efficiencies, even during brief 1, or 3-hour exposures. In an effort to extend these in vitro findings targeting a mixed leukocyte population and explore the feasibility in vivo, we next performed repopulation experiments in myeloablated murine recipients. Unseparated marrow cells harvested from donor animals were depleted of red blood cells, washed and immediately transduced on fibronectin fragment in the presence of murine stem cell factor. Following a 1 hour exposure to lentivector (VSV-G/RRLsin-cPPThPGK-EGFPwpre), cells were washed repeatedly, resuspended and injected into myeloablated recipients (n=10). Animals showed ready hematopoietic reconstitution and demonstrated average GFP marking of 31% (range: 17–41.2%) in peripheral blood 20 weeks after transplantation. Gene marking in secondary recipients 9 weeks after reconstitution (n=15, 3 recipient animals per donor) persisted at 29% on average (range 14.9–66%). Results also demonstrate transduction of granulocytes, B- and T-lymphocytes, as well as stable long-term GFP expression in primary and secondary animals. Copy number determination by real-time PCR in marrow cells from primary recipients shows an average of 4 proviral copies (range 2.1–8.1) per GFP-expressing cell. Our studies confirm that HIV-derived lentivirus vectors are ideally suited for the transduction of murine long-term repopulating cells. We hypothesize that ultra-short transduction actively preserves stem cell content in the inoculum. Moreover, this protocol represents an ideal platform for subsequent in vivo selection to achieve complete phenotype correction and high-level therapeutic chimerism required for some applications. We anticipate that our strategy may prove particularly useful in situations where the target stem cell quantity is greatly limited and cells are of poor ex vivo viability.

Effects of cell cycle activation on the short-term engraftment properties of ex vivo expanded murine hematopoietic cells

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2829-2837 ◽  
Author(s):  
Stephen J. Szilvassy ◽  
Todd E. Meyerrose ◽  
Barry Grimes
Keyword(s):  
Cell Cycle ◽  
Stem Cell ◽  
Clinical Utility ◽  
Cell Function ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Short Term ◽  
Hematopoietic Stem

Loss of long-term hematopoietic stem cell function in vitro is associated with cell cycle progression. To determine whether cytokine-induced proliferation also limits the rate of short-term engraftment and potential clinical utility of ex vivo expanded hematopoietic cells, murine Sca-1+c-kit+Lin− cells were cultured in interleukin-6 (IL-6), IL-11, granulocyte colony-stimulating factor (G-CSF), stem cell factor, flk-2 ligand, and thrombopoietin for 7 days. Cells amplified 2000-fold were then stained with Hoechst 33342, separated into G0/G1 (72% ± 3%) or S/G2/M (27% ± 3%) fractions by flow sorting, and injected into lethally irradiated mice. Although long-term (more than 6 months) engraftment of lymphoid and myeloid lineages was greater in primary and secondary recipients of expanded cells residing in G0/G1 at the time of transplantation, there were no noted differences in the short-term (less than 6 weeks) recovery kinetics of circulating blood cells. When hematopoietic cells were expanded in cultures containing the tetrapeptide stem cell inhibitor N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) to reduce progenitor cycling prior to transplantation, again there were no differences observed in short-term reconstitution by inhibited or uninhibited cells. Interestingly, AcSDKP significantly accelerated engraftment by expanded hematopoietic cells when administered in vivo at the time of transplantation. Leukocytes recovered to 20% of normal levels approximately 1 week faster, and thrombocytopenia was largely abrogated in AcSDKP-treated versus untreated mice. Therefore, while AcSDKP can accelerate the engraftment of ex vivo expanded hematopoietic progenitors, which suggests a relatively simple approach to improve their clinical utility, its effects appear unrelated to cell cycle arrest.

scholarly journals Structurally Specific Heparan Sulfates Support Primitive Human Hematopoiesis by Formation of a Multimolecular Stem Cell Niche

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4641-4651 ◽  
Author(s):  
Pankaj Gupta ◽  
Theodore R. Oegema ◽  
Joseph J. Brazil ◽  
Arkadiusz Z. Dudek ◽  
Arne Slungaard ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Line ◽  
Stem Cell Niche ◽  
Ex Vivo ◽  
Recent Observation ◽  
Hematopoietic Progenitors ◽  
Platelet Factor ◽  
Cell Niche

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P < .005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34+/HLA-DR−cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34+ cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.

Safety outcomes and long-term effectiveness of ex vivo autologous cultured limbal epithelial transplantation for limbal stem cell deficiency

2016 ◽  
Vol 101 (5) ◽  
pp. 640-649 ◽  
Author(s):  
Adriano Fasolo ◽  
Emilio Pedrotti ◽  
Mattia Passilongo ◽  
Giorgio Marchini ◽  
Cristina Monterosso ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Limbal Stem Cell Deficiency ◽  
Safety Outcomes ◽  
Limbal Stem Cell

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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