Stem Cells Defects in Systemic Sclerosis.

Abstract Systemic Sclerosis (SSc) is a connective tissue disease characterized by early generalized microangiopathy and culminating in systemic fibrosis. Recent studies have provided evidence that SSc is associated with a reactive but ineffective angiogenesis, so that the disease finally leads to the irreversible loss of capillaries. Aim of the study was to investigate whether impaired vasculogenesis in SSc is due to defective characteristics in BM microenvironment. Peripheral blood (PB) samples were collected from 70 patients (pts): circulating endothelial progenitors (CEPs) were characterized as CD45−/CD133+ and evaluated by flow cytometry. BM samples were collected from 14 SSc pts and hematopoiesis evaluated by various assays. CD133+ cells were isolated by immunomagnetic sorting (IMS) and grown in order to induce endothelial differentiation. Long-term bone marrow cultures (LTBMC) were assessed and the number of stromal clonogenic precursors evaluated by a CFU-F (colony-forming unit fibroblast) assay. Mesenchymal stem cells (MSC) were separated by IMS for the expression of the nerve growth factor-receptor (NGF-R+) and grown in order to assess the clonogenic potential and the proliferative capacity, while their multipotential differentiation ability was determined after culture in different conditioned media. Phenotypic analysis of BM mononuclear cells showed a greater expression of the surface markers P1H12 and CD105 TGF-β receptor (1.2%±0.6 vs 0.5%±0.1 in normal controls, p=0.01 and 9.9%±5 vs 4.7%±3, p=0.02 respectively), but lower percentages of NGF-R+ stromal cell precursors (0.73±0.5 vs 1.61±0.6, p=0.02) and CD133+ cells (0.36%±0.4 vs 1.2%±0.8, p=0.05). On the contrary, the absolute number of CEPs in PB was higher in patients with SSc than in healthy controls (mean values 2.1 cells/μL vs 0.26 cells/μL, p=0.04). When BM CD133+ cells were grown in the presence of VEGF, only 3/12 cases gave endothelial differentiation, but always with a reduced proliferative ability. All pts showed a defective stromal compartment and a reduced number of BM stromal precursors, as detected by the LTBMC and by the lower CFU-F frequency (4%±3.2 vs 43%±19.8/1x10(e)6 LDMNCs, p=0.002 and 7±12.8 vs 69±61/1x10(e)5 NGF-R+ cells, p=0.01). Interestingly, NGF-R+ MSC overexpressed KDR and CD117 (26.4%±7.4 vs 4.6%±1.7, p=0.01 and 87.7%±5.1 vs 57.6%±11, p=0.03 respectively): when grown in the presence of VEGF they gave rise to endothelial colonies, only in 2/8 cases they formed a confluent layer with fibroblastic morphology but a reduced proliferative ability, while in the presence of adipogenic or osteogenic inductive media they failed to origin specific differentiation. Moreover, all “in vitro” differentiated endothelial cells even before activation showed high levels of CD62-E, VCAM-1 and CD105 expression, suggestive of the presence of increased levels of proangiogenic factors in BM. The results of this study provide evidence that patients with SSc have a stem cell defect involving both the hematopoietic and the stromal cells compartments. The higher expression of KDR on NGF-R+ cells suggests a role for VEGF in inducing endothelial differentiation of MSC, so resulting in a depletion of stromal precursors. The continuous recruitment of endothelial progenitors to sites of vascular injury, suggested by the high numbers of CEPs in PB, might lead to the irreversible BM damage we observed.

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Phenotypical and functional characteristics of in vitro expanded bone marrow mesenchymal stem cells from patients with systemic sclerosis

Annals of the Rheumatic Diseases ◽

10.1136/ard.2007.071233 ◽

2007 ◽

Vol 67 (4) ◽

pp. 443-449 ◽

Cited By ~ 73

Author(s):

J Larghero ◽

D Farge ◽

A Braccini ◽

S Lecourt ◽

A Scherberich ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Systemic Sclerosis ◽

Autoimmune Disease ◽

Healthy Donor ◽

Large Scale ◽

Mononuclear Cells ◽

Differentiation Potential

Background:Mesenchymal stem cells (MSCs) have a potential immunomodulatory role in autoimmune disease; however, the qualitative properties and haematopoietic support capacity of MSCs derived from patients with autoimmune disease is unclear.Objectives:To further characterise phenotypically and functionally bone marrow (BM)-derived MSCs from patients with systemic sclerosis (SSc).Methods:Key parameters of BM-derived MSC function and phenotype were assessed in 12 patients with SSc and compared with 13 healthy normal controls. The parameters included the ability to: form colony-forming unit fibroblasts (CFU-F), differentiate along the adipogenic and osteogenic lineages, express cell surface antigens defining the MSCs population, support normal haematopoiesis and suppress in vitro lymphocyte proliferation induced by either anti-CD3∊ plus anti-CD28 monoclonal antibodies or the mixed lymphocyte reaction.Results:SSc MSCs were shown to have a similar characteristic phenotype, capacities to form CFU-F and to differentiate along adipogenic and osteogenic lineages as those of healthy donor MSCs. The ability of SSc MSCs to support long-term haematopoiesis was also identical to that of controls. Both healthy donor and SSc BM MSCs reduced the proliferation of autologous and allogeneic peripheral blood mononuclear cells in a cell number dependent fashion.Conclusions:These results show that BM-derived MSCs from patients with SSc under the described culture conditions exhibit the same phenotypic, proliferative, differentiation potential and immunosuppressive properties as their healthy counterparts and could therefore be considered in an autologous setting. Further studies are needed to ensure the quality and safety of large-scale expansion of patient MSCs prior to their potential use in clinical trials.

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Abstract 697: Mobilization of Oct-4 + Bone Marrow-Derived Very Small Embryonic-Like Stem Cells in Patients with Acute Myocardial Infarction

Circulation ◽

10.1161/circ.116.suppl_16.ii_130-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Wojciech Wojakowski ◽

Magda Kucia ◽

Boguslaw Machalinski ◽

Edyta Paczkowska ◽

Joanna Ciosek ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Stem Cells ◽

Bone Marrow ◽

Pluripotent Stem Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Small Cells ◽

Acute Mi

Bone marrow-derived CD34 + CXCR4 + progenitor cells are mobilized into peripheral blood early in acute myocardial infarction (MI). Adult murine bone marrow contains population of small CD34 + lin − CD45 − CXCR4 + cells expressing markers of pluripotent stem cells (PSC) SSEA, Oct-4 and Nanog. This population of very small embryonic-like cells (VSEL) has unique morphology (small size 2– 4 μm, large nucleus, euchromatin) and capability to form embrioid bodies (EB). Murine EB-derived cells can in vitro differentiate into cells from all three germ layers including cardiomyocytes. We hypothesized that in patients with acute MI small cells expressing the VSEL immunophenotype and PSC markers are present in bone marrow and mobilized into peripheral blood. Blood samples (20 mL) from 18 patients with acute MI were obtained after 12 hours, 2 and 5 days after symptoms onset. Bone marrow samples (20 mL) were obtained from 2 patients with acute MI and 3 healthy volunteers. Mononuclear cells were isolated using hypotonic lysis and samples were analyzed by FACS. Mobilization of following cell populations was confirmed: hematopoietic lin − CD45 + CXCR4 + , lin − CD45 + CD133 + , lin − CD45 + CD34 + and non-hematopoietic (VSEL) lin − CD45 − CXCR4 + , lin − CD45 − CD133 + , lin − CD45 − CD34 + . Analysis of the cell number using lymphocyte gate showed more significant increase of CD45 + (hematopoietic) populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells. After gating for small events (VSEL size range) we found more significant mobilization of small, non-hematopoietic populations of lin − CD34 + , lin − CD133 + and lin − CXCR4 + cells (Table ). The expression of PSC markers (Oct-4, Nanog, SSEA-1) in VSEL was confirmed using real-time RT-PCR. Conclusion: We report for the first time that acute MI is associated with mobilization of non-hematopoietic VSELs expressing pluripotent stem cells markers.

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Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo

Blood ◽

10.1182/blood.v88.11.4102.4102 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4102-4109 ◽

Cited By ~ 8

Author(s):

CI Civin ◽

G Almeida-Porada ◽

MJ Lee ◽

J Olweus ◽

LW Terstappen ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Population ◽

Mononuclear Cells ◽

Cell Subset ◽

Fetal Sheep ◽

Adult Human

Abstract Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38-cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38-subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long- term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38-cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

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Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement

Stem Cells International ◽

10.1155/2018/7396905 ◽

2018 ◽

Vol 2018 ◽

pp. 1-17 ◽

Cited By ~ 30

Author(s):

Sushrut Dakhore ◽

Bhavana Nayer ◽

Kouichi Hasegawa

Keyword(s):

Stem Cells ◽

Cell Culture ◽

Pluripotent Stem Cells ◽

Large Scale ◽

Embryonic Stem ◽

Current Status ◽

Specific Cell ◽

Therapeutic Applications ◽

Proliferative Ability

Over the past two decades, human embryonic stem cells (hESCs) have gained attention due to their pluripotent and proliferative ability which enables production of almost all cell types in the human body in vitro and makes them an excellent tool to study human embryogenesis and disease, as well as for drug discovery and cell transplantation therapies. Discovery of human-induced pluripotent stem cells (hiPSCs) further expanded therapeutic applications of human pluripotent stem cells (PSCs). hPSCs provide a stable and unlimited original cell source for producing suitable cells and tissues for downstream applications. Therefore, engineering the environment in which these cells are grown, for stable and quality-controlled hPSC maintenance and production, is one of the key factors governing the success of these applications. hPSCs are maintained in a particular niche using specific cell culture components. Ideally, the culture should be free of xenobiotic components to render hPSCs suitable for therapeutic applications. Substantial efforts have been put to identify effective components, and develop culture conditions and protocols, for their large-scale expansion without compromising on quality. In this review, we discuss different media, their components and functions, including specific requirements to maintain the pluripotent and proliferative ability of hPSCs. Understanding the role of culture components would enable the development of appropriate conditions to promote large-scale, quality-controlled expansion of hPSCs thereby increasing their potential applications.

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Functional and phenotypic analysis of in vitro stimulated canine peripheral blood mononuclear cells

Veterinary Immunology and Immunopathology ◽

10.1016/0165-2427(94)05330-u ◽

1995 ◽

Vol 45 (1-2) ◽

pp. 55-71 ◽

Cited By ~ 11

Author(s):

Ariel L. Rivas ◽

Edward S. Kimball ◽

Fred W. Quimby ◽

Douglas Gebhard

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Phenotypic Analysis ◽

Peripheral Blood Mononuclear ◽

Blood Mononuclear Cells

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Donor Origin of Multipotent Adult Progenitor Cells in Radiation Chimeras.

Blood ◽

10.1182/blood.v106.11.1395.1395 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1395-1395

Author(s):

Morayma Reyes ◽

Jeffrey S. Chamberlain

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Y Chromosome ◽

Stromal Cells ◽

Mononuclear Cells ◽

Multipotent Adult Progenitor Cells

Abstract Multipotent Adult Progenitor Cells (MAPC) are bone marrow derived stem cells that can be extensively expanded in vitro and can differentiate in vivo and in vitro into cells of all three germinal layers: ectoderm, mesoderm, endoderm. The origin of MAPC within bone marrow (BM) is unknown. MAPC are believed to be derived from the BM stroma compartment as they are isolated within the adherent cell component. Numerous studies of bone marrow chimeras in human and mouse point to a host origin of bone marrow stromal cells, including mesenchymal stem cells. We report here that following syngeneic bone marrow transplants into lethally irradiated C57Bl/6 mice, MAPC are of donor origin. When MAPC were isolated from BM chimeras (n=12, 4–12 weeks post-syngeneic BM transplant from a transgenic mouse ubiquitously expressing GFP), a mixture of large and small GFP-positive and GFP-negative cells were seen early in culture. While the large cells stained positive for stroma cell markers (smooth muscle actin), mesenchymal stem cell makers (CD73, CD105, CD44) or macrophages (CD45, CD14), the small cells were negative for all these markers and after 30 cell doublings, these cells displayed the classical phenotype of MAPC (CD45−,CD105−, CD44−, CD73−, FLK-1+(vascular endothelial growth factor receptor 2, VEGFR2), Sca-1+,CD13+). In a second experiment, BM obtained one month post BM transplant (n=3) was harvested and mononuclear cells were sorted as GFP-positive and GFP-negative cells and were cultured in MAPC expansion medium. MAPC grew from the GFP-positive fraction. These GFP positive cells displayed the typical MAPC-like immunophenotypes, displayed a normal diploid karyotype and were expanded for more than 50 cell doublings and differentiated into endothelial cells, hepatocytes and neurons. To rule out the possibility that MAPC are the product of cell fusion between a host and a donor cell either in vivo or in our in vitro culture conditions, we performed sex mismatched transplants of female GFP donor BM cells into a male host. BM from 5 chimeras were harvested 4 weeks after transplant and MAPC cultures were established. MAPC colonies were then sorted as GFP-positive and GFP- negative and analyzed for the presence of Y-chromosome by FISH analysis. As expected all GFP-negative (host cells) contained the Y-chromosome whereas all GFP-positive cells (donor cells) were negative for the Y-chromosome by FISH. This proves that MAPC are not derived from an in vitro or in vivo fusion event. In a third study, BM mononuclear cells from mice that had been previously BM-transplanted with syngeneic GFP-positive donors (n=3) were transplanted into a second set of syngeneic recipients (n=9). Two months after the second transplant, BM was harvested and mononuclear cells were cultured in MAPC medium. The secondary recipients also contained GFP-positive MAPC. This is the first demonstration that BM transplantation leads to the transfer of cells that upon isolation in vitro generate MAPCs and, whatever the identity of this cell may be, is eliminated by irradiation. We believe this is an important observation as MAPC hold great clinical potential for stem cell and/or gene therapy and, thus, BM transplant may serve as a way to deliver and reconstitute the MAPC population. In addition, this study provides insight into the nature of MAPC. The capacity to be transplantable within unfractionated BM transplant renders a functional and physiological distinction between MAPC and BM stromal cells. This study validates the use of unfractionated BM transplants to study the nature and possible in vivo role of MAPC in the BM.

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A Novel Population of Oct-4+ SSEA-1+ CXCR4+ CD34+ CD133+ Lin− CD45− Very Small Embryonic-Like (VSEL) Stem Cells Identified in Human Cord Blood.

Blood ◽

10.1182/blood.v108.11.3195.3195 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3195-3195

Author(s):

Magda Kucia ◽

Maciej Halasa ◽

Marcin Wysoczynski ◽

Magda Baskiewicz-Masiuk ◽

Ewa Zuba-Surma ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cord Blood ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Specific Cell ◽

Human Cord Blood ◽

Organ Tissue ◽

Hypotonic Lysis

Abstract Mononuclear cells (MNC) isolated from bone marrow (BM) or cord blood (CB) contributes to organ/tissue regeneration, however, the identity of the specific cell type(s) involved remains unknown. Recently we identified in murine BM a homogenous population of rare (~0.01% of BM MNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1, highly express Rif-1 telomerase protein and display several features typical for primary embryonic stem cells such as a small size (~2–4 um in diameter), a large nuclei surrounded by a narrow rim of cytoplasm, and open-type chromatin (euchromatin) that is typical for embryonic stem cells (Leukemia2006;20,857–869). These cells were named very small embryonic-like (VSELs) stem cells. We will present a new two step isolation procedure to purify a similar population of cells from human CB, which is based on isolation of CB mononuclear cells (CB MNC) by hypotonic lysis and multiparameter FACS sorting. Accordingly, we perform hypotonic lysis of CB to remove erythrocytes and to enrich for CB MNC combined with multiparameter sorting for CXCR4+AC133+CD34+lin−CD45− CB MNC. CB-derived VSELs (CB-VSELs) isolated this way similarly as those isolated from adult murine BM are very small (3–5 um), possess large nuclei containing unorganized euchromatin, express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. In vitro cultures CB-VSELs are able to grow neurospheres that gave rise to neuronal lineages (beta-III tubulin+, nestin+, O4+, MBP+, GFAP+) and cardiomyocytes (beta-myosin heavy chain+, alpha-sarcomeric actin. Based on this we conclude that CB contains VSELs and that the majority of these CB VSELs are lost during routine procedures employed currently for banking of CB MNC. Thus based on our observations, new more efficient methods of CB banking are needed that will enrich/preserve these cells in CB units during preparation before storage. Furthermore, we conclude that CB tissue/organ regenerating potential may be much higher than initially postulated if the proper fraction of CB MNC is employed and we are currently testing this hypothesis in animal models.

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Marked Improvement of Skin Involvement in Severe Progressive Systemic Sclerosis after Transplantation of Mesenchymal Stem Cells from an Allogeneic Haploidentical Related Donor.

Blood ◽

10.1182/blood.v110.11.4865.4865 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4865-4865

Author(s):

Maximilian Christopeit ◽

Marit Schendel ◽

Lutz Peter Mueller ◽

Gernot Keyszer ◽

Gerhard Behre

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Systemic Sclerosis ◽

Renal Involvement ◽

Progressive Systemic Sclerosis ◽

In Vitro Expansion ◽

Modified Rodnan Skin Score ◽

Diffuse Cutaneous Systemic Sclerosis ◽

Related Donor

Abstract Introduction. Mesenchymal stem cells (MSC) show immunosuppressive capacity in an allogeneic host. Severe progressive systemic sclerosis (SSc) is an autoimmune disease in which prognosis is worsened in individuals presenting with diffuse cutaneous, lung and renal involvement. Immunosuppression can improve the course of the disease. Methods. We transplanted a 41 year old female patient suffering from diffuse cutaneous systemic sclerosis with MSC from her father after in vitro expansion of the MSC. Results. The patient presented with a remarkable clinical response. Her ulcer surface decreased, the 17 Site Modified Rodnan Skin Score, vascular ultrasound, mouth aperture, the Visual Analogue Scale for pain (VAS), the Valentini Activity Score and the Hannover Functional Questionnaire showed encouraging improvements. The patient did not experience any adverse events during the first 300 days after transplantation. Conclusions. The transplantation of MSC from a haploidentical donor into a patient with systemic sclerosis is feasible, safe and effective. Furthermore is the transplantation of MSC from a haploidentical donor into an adult without severe pharmacological immunosuppression at the time of transplantation feasible and safe.

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