scholarly journals Growth and Differentiation of Circulating Stem Cells After Extensive Ex Vivo Expansion

2021 ◽  
Author(s):  
Silvia Barbon ◽  
Senthilkumar Rajendran ◽  
Thomas Bertalot ◽  
Monica Piccione ◽  
Marco Gasparella ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Muscle Damage ◽  
Adult Stem Cells ◽  
Ex Vivo ◽  
Myogenic Differentiation ◽  
Calcium Flux ◽  
Morphological Studies

Abstract Background: Stem cell therapy is gaining momentum as an effective treatment strategy for degenerative diseases. Adult stem cells isolated from various sources (i.e., cord blood, bone marrow, adipose tissue) are being considered as a realistic option due to their well-documented therapeutic potentials. Our previous studies standardized a method to isolate circulating multipotent cells (CMCs) that are able to sustain long term in vitro culture and differentiate towards mesodermal lineages. Methods: In this work, long-term cultures of CMCs were stimulated to study in vitro neuronal and myogenic differentiation. After induction, cells were analysed at different time points. Morphological studies were performed by scanning electron microscopy and specific neuronal and myogenic marker expression were evaluated using RT-PCR, flow cytometry and western blot. For myogenic plasticity study, CMCs were transplanted into in vivo model of chemically-induced muscle damage. Results: After neurogenic induction, CMCs showed characteristic dendrite-like morphology and expressed specific neuronal markers both at mRNA and protein level. The calcium flux activity of CMCs under stimulation with potassium chloride and the secretion of noradrenalin confirmed their ability to acquire a functional phenotype. In parallel, the myogenic potential of CMCs was confirmed by their ability to form syncytium-like structures in vitro and express myogenic markers both at early and late phases of differentiation. Interestingly, in a rat model of bupivacaine-induced muscle damage, CMCs integrated within the host tissue taking part in tissue repair. Conclusion: Overall, collected data demonstrated long-term cultured CMCs retain proliferative and differentiative potentials suggesting to be a good candidate for cell therapy.

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.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

scholarly journals “Opening” The Mesenchymal Stem Cell Tool Box

2009 ◽  
Vol 03 (03) ◽  
pp. 240-249 ◽  
Author(s):  
Fares Zeidán-Chuliá ◽  
Mami Noda
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Current Status ◽  
Tooth Replacement ◽  
Cell Based Therapy ◽  
Adult Mesenchymal Stem Cells ◽  
Wide Potential ◽  
New Strategies

ABSTRACTAdult mesenchymal stem cells (MSCs) are adherent stromal cells able to self-renew and differentiate into a wide variety of cells and tissues. MSCs can be obtained from distinct tissue sources and have turned out to be successfully manipulated in vitro. As adult stem cells, MSCs are less tumorigenic than their embryonic correlatives and posses another unique characteristic which is their almost null immunogenicity. Moreover, these cells seem to be immunosuppressive in vitro. These facts together with others became MSCs a promising subject of study for future approaches in bioengineering and cell-based therapy. On the other hand, new strategies to achieve long-term integration as well as efficient differentiation of these cells at the area of the lesion are still challenging, and the signalling pathways ruling these processes are not completely well characterized. In this review, we are going summarize the general landscape and current status of the MSC tool as well as their wide potential in tissue engineering, from neuronal to tooth replacement. Highlights and pitfalls for further clinical applications will be discussed. (Eur J Dent 2009;3:240-249)

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

scholarly journals Efficient Ex Vivo Expansion of Highly Purified Human Umbilical Cord Blood-Derived Very Small CD34+lin-CD45- Stem Cells into Functional Endothelial Cells in Vitro in Chemically Identified, Feeder Layer-Free Medium Supplemented with Nicotinamide

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4882-4882
Author(s):  
Alison Domingues ◽  
Kamila Bujko ◽  
Magdalena Kucia ◽  
Janina Ratajczak ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Cell Therapy ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Germ Layer ◽  
Differentiation Factors

Background . There is an ongoing search for multipotent stem cells from umbilical cord blood (UCB) with trans-germ layer differentiation potential that can be employed in repairing damaged organs and also expanded into transplantable hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). The existence of such cells in postnatal life could also revive the concept of hemangioblasts or hemangioblast-like cells in adult hematopoietic organs. Our group was the first to isolate a population of small CD34+CD133+lin-CD45- early-development stem cells from human hematopoietic tissues, including UCB. Based on the validated expression of early-development markers, these cells were named "very small embryonic-like stem cells" (VSELs, Circulation Res 2019; 124:208-210). Currently, more than 25 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and demonstrated their in vivo contribution to all three germ layer lineages. Thus, VSELs could be very useful in regenerative medicine in the field of angiogenesis, and UCB is an attractive source, with easy accessibility and tolerance to allogenic grafts. However, the low number of these cells in UCB and their quiescence are limiting factors. Therefore, in vitro differentiation of VSELs into endothelial progenitor cells (EPCs) would allow improvement in the ability to expand endothelial cells and could represent a clinically relevant alternative to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) for cell therapy without ethical problems and undesirable side effects. Hypothesis. We hypothesized that UCB-purified, very small, early-developmentCD34+lin-CD45-stem cells can be ex vivo expanded into functional EPCs. Materials and Methods. VSELs highly purified by FACS were expanded into EPCs in pro-angiogenic medium supplemented with mesodermic differentiation factors and then endothelial differentiation factors in the presence of nicotinamide and UM171. In parallel, we expanded EPCs from MNCs isolated from the same UCB units by employing a classical protocol (Methods in Enzymology 2008, 445:303-29). The EPC nature of the expanded VSEL-derived cells was confirmed by the expression of typical EPC markers as well as by in vitro angiogenic assays. Results. Our differentiation cocktail allowed us to differentiate and expand VSELs into EPCs. In our expansion medium (Figure 1), the very small, round VSELs smaller than 6 mm in diameter proliferated and differentaited over time into larger and extended cells with a cobblestone morphology similar to the EPC control cells, and we confirmed their endothelial characteristics by cytometry analysis. Like EPCs, VSEL-derived EPCs were positive for CD31, CD144, KDR, and CD105 and negative for mesenchymal surface markers, such as CD90. They also performed similarly to EPCs in classical vasculogenic tests, including adhesion, proliferation, migration, and tubulogenesis assays. Conclusions. This work shows, for the first time, efficient VSEL differentiation into functional endothelial cells with vasculogenic properties without the help of co-culture over feeder-layers or viral vectors in medium supplemented with nicotinamide and UM171. These findings allow us to propose these cells as an interesting cell therapy product. These results also reopen the question of the existence of hemangioblast-like cells in postnatal tissues. We are currently testing these cells in vivo in model of hind limb ischemia. Figure 1 Disclosures No relevant conflicts of interest to declare.

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.

Therapeutic potential of transdifferentiated cells

2005 ◽  
Vol 108 (4) ◽  
pp. 309-321 ◽  
Author(s):  
Zoë D. BURKE ◽  
David TOSH
Keyword(s):  
Stem Cells ◽  
Cell Therapy ◽  
Adult Stem Cells ◽  
Therapeutic Potential ◽  
Cell Types ◽  
Therapeutic Modality ◽  
Differentiated Cells ◽  
Degenerative Disorders ◽  
Clinical Conditions

Cell therapy means treating diseases with the body's own cells. The ability to produce differentiated cell types at will offers a compelling new approach to cell therapy and therefore for the treatment and cure of a plethora of clinical conditions, including diabetes, Parkinson's disease and cardiovascular disease. Until recently, it was thought that differentiated cells could only be produced from embryonic or adult stem cells. Although the results from stem cell studies have been encouraging, perhaps the most startling findings have been the recent observations that differentiated cell types can transdifferentiate (or convert) into a completely different phenotype. Harnessing transdifferentiated cells as a therapeutic modality will complement the use of embryonic and adult stem cells in the treatment of degenerative disorders. In this review, we will examine some examples of transdifferentiation, describe the theoretical and practical issues involved in transdifferentiation research and comment on the long-term therapeutic possibilities.

scholarly journals Modulation of Aplnr signaling is required during the development and maintenance of the hematopoietic system

2020 ◽  
Author(s):  
Melany Jackson ◽  
Antonella Fidanza ◽  
A. Helen Taylor ◽  
Stanislav Rybtsov ◽  
Richard Axton ◽  
...  
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Null Mouse ◽  
List Type ◽  
Hematopoietic Stem ◽  
Apelin Receptor

ABSTRACTApelin receptor (Aplnr/Agtrl1/Apj) marks a transient cell population during the differentiation of hematopoietic progenitor cells (HPCs) from pluripotent stem cells but the function of this signalling pathway during hematopoietic development both in vitro and in vitro is poorly understood. We generated an Aplnr-null mouse embryonic stem cell (mESC) line and demonstrated that they are significantly impaired in the production of HPCs indicating that the Aplnr pathway is required for their formation. Using Aplnr-tdTomato reporter mESCs we demonstrated that is expressed in a population of differentiating mesodermal cells committed to a hematopoietic and endothelial fate. Activation of this signaling pathway by the addition of the Apelin ligand to differentiating ESCs has no effect on the production of HPCs but the addition to ex vivo AGM cultures impaired the generation of long term reconstituting hematopoietic stem cells and appeared to drive myeloid differentiation. Taken together, our data suggest that the Aplnr pathway is required for the generation of cells that give rise to HSCs during development but its subsequent down regulation is required for their maintenance.HIGHLIGHTSHematopoietic differentiation is impaired in Aplnr-null ESCsAplnr-tdTomato reporter marks a subpopulation of ESC-derived mesoderm.Aplnr signaling drives the maturation of lineage-committed myeloid progenitorsIn AGM explant cultures HSC activity is reduced in the presence of Aplnr ligands.

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