CD34 Antibody - an overview (2023)

A novel CD34 antibody was used that enriches for enhanced colony forming cells and efficiently detects HSCs/ovine progenitors in umbilical cord blood, BM, mobilized peripheral blood [78].

Of:Perinatal Stem Cells, 2018

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Developmental Biology of Stem Cells

Momoko Yoshimoto, Mervin C. Yoder, euFetal and Neonatal Physiology (Fifth Edition), 2017

Isolation of human hematopoietic stem cells and the human hematopoietic hierarchy system

InhumanCD34 antibodywas first described to enrich human myeloid progenitor cells from bone marrow cells.87Since then, human CD34 has been used to isolate HSCs in experimental and clinical settings.Figure 114-2). In single-cell transplantation into the bone marrow of immunodeficient female mice, Notta et al.88identified CD34+CD38CD90+CD45RACD49f+cells as long-term human HSCs with multilineage engraftment, while CD34+CD38CD90CD45RACD49f-the cells are multipotent progenitor cells that engraft in short-term immunodeficient mice. CD34the cells have also been reported to contain repopulated SCID cells, and a recent report has shown that CD34CD38CD93altocells are self-renewing and can be placed on CD34+cells in the hematopoietic hierarchy.82However, the frequency of CD34HSCs are much lower than for CD34+HSCs and over 99% of human HSCs must be CD34+.27

In the classic model of hematopoiesis, long-term HSCs are positioned at the top of the hematopoietic hierarchy, followed by short-term HSCs and multipotent stem cells (seeFigure 114-2). After this point, myeloid and lymphoid cells differentiate as common myeloid progenitors and common lymphoid progenitors. Common myeloid progenitors produce all cells of the myeloid lineage, whereas common lymphoid progenitors produce only lymphoid cells. In the mouse model, the system is not so simple, and myeloid potential is also demonstrated in early lymphoid progenitors, as shown inFigure 114-1.89-91However, it is still unknown whether the human hematopoiesis system follows the classical model (seeFigure 114-2). Doulatov is colleagues92investigated the developmental potential of seven adult cord blood and bone marrow progenitor populations by in vitro clonal assay, providing evidence that CD34+CD38CD90neg-loCD45RA+human multilymphoid stem cells give rise to all lymphoid cells as well as myeloid cells such as monocytes, macrophages, and dendritic cells. This indicates that human hematopoiesis does not follow a classic model of myeloid-lymphoid segregation.92,93

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Intravascular stents seeded with endothelial stem cells (EPC)

C. Zhu, euHeart regeneration and repair, 2014

5.3.2Strategy for EPC-seeded intravascular stents

EPCs can express several specific surface markers such as VEGFR, CD34 and CD133. Currently, the development of third-generation intravascular stents is largely focused on intravascular stents coated with specific surface marker antibodies to selectively capture EPCs. cow printand others(2003) was the first to propose the pioneering strategy of coating stents with mouse anti-human monoclonal.CD34 antibodyto capture EPCs from the blood stream. Clinical results have shown that after implantation, intravascular stents can rapidly capture EPCs from the patient's circulation and allow a monolayer of endothelial cells to coat the stents within 2 days (ongand others, 2005). Rapid surface endothelialization effectively inhibited thrombus formation and restenosis and greatly reduced the required duration of treatment with drugs such as clopidogrel in patients after surgery. Results of clinical trials published in 2010 indicated that the incidence of thrombosis was significantly reduced compared to the first two generations of intravascular stents in patients over 70 years of age, indicating that implantation of stents coated with CD34 antibody is a safe therapeutic strategy. and effective for intravascular stenting, especially in elderly patients with coronary artery disease (Azzarelliand others, 2010). Researchers have also developed several similar stents, such as the CD133 bioengineered stents and the VEGF bioengineered stents, which have been shown to reduce the incidence of thrombosis and promote long-term patency of blood vessels.

Currently, this type of bioengineered stent has been approved by Conformité Européene (CE) and entered the market in more than 60 countries worldwide. However, these stents have not been approved by the US FDA or the China State Food and Drug Administration (SFDA) because there is still controversy over their safety and effectiveness. Recent studies have shown that only 0.4 ± 0.2% of CD34+cells are EPCs (expressing VEGFR-2 and CD133) in the blood, which means that only one in 250 CD34+cells is an EPC. Therefore, the CD34 antibody can capture not only EPCs, but also large numbers of stellate cells and hematopoietic progenitor cells (including EPCs, myeloid progenitor cells, and lymphoid progenitor cells). These competing cells (such as platelets, lymphoid cells, and monocytes), whose concentrations in the blood are significantly higher than those of EPCs, can bind rapidly to CD34 antibodies on intravascular stents and coat the captured EPCs, resulting in failure of the intravascular stent technique. coated with CD34 antibody (Wendeland others,2010).

VEGFR-2 is the major receptor for VEGF, which is expressed on EPCs, hematopoietic progenitor cells and vascular endothelial cells, and regulates endothelial cell growth, survival, migration and permeability, as well as the process of vascular tube formation. VEGFR also mediates the signaling cascade pathway, which plays a central regulatory role in the process of angiogenesis. In 2008, Markwayand othersVEGFR-2 antibodies immobilized on the stent surface; however, this stent is similar to the CD34 antibody-coated stent and also captures lymphoid progenitor cells, myeloid progenitor cells that can differentiate intolymphocytes and other cells, when it captures EPCs and endothelial cellsdirectrepair of damaged blood vessels (Markwayand others,2008). Human CD133 was first discovered byyinand others(1997)of CD34+hematopoietic stem cells through the artificial separation of CD133 monoclonal antibodies. CD133 has greater specificity for EPCs than CD34 and is expressed primarily on early hematopoietic stem cells, pluripotent hematopoietic stem cells, and blood vessel stem cells. Therefore, the clinical effect of CD133 antibody coated stents is better than CD34 antibody coated stents. The specificity of EPC capture is still unresolved.

To solve this problem,Nakazawaand others(2010)immobilized an anti-human CD34 antibody on the surface of commercially available sirolimus-eluting stents and evaluated the differences in surface endothelialization between bare-metal stents, anti-CD34 antibody stents, and sirolimus-eluting stents in plain and overlapping stents. The result showed that application of anti-CD34 antibody on the surface of sirolimus-eluting stents improved re-endothelialization compared to sirolimus-eluting stents alone after 3 and 14 days. However, anti-CD34 sirolimus resulted in decreased endothelialization compared to stents containing anti-CD34 antibody alone at 3 days (sirolimus-anti-CD34 36 ± 26% and anti-CD34 76 ± 8%) and 14 days (sirolimus-anti-CD34). anti) - CD34 82 ± 8% and anti-CD34 98 ± 2%) (Nakazawa,and others2010). The main reason for this is that the eluting drug can inhibit the growth of endothelial cells and EPCs, while inhibiting the proliferation of smooth muscle cells.

(Video) CD34

Therefore, the success of EPC-seeded intravascular stents largely depends on the selection of the optimal target on the surface of the EPCs. An ideal capture factor should have high affinity and high specificity for EPCs. Ideally, this factor would also have anti-inflammatory and antithrombotic functions. In recent years, researchers have used several methods to seed EPCs on the stent surface. The first method is the use of specific antibodies against EPCs, such as the aforementioned antibody CD34, and antibodies with even greater specificity, such as against CD133 and VEGFR-2. However, none of these antibodies have the ability to selectively capture EPCs (Wendeland others,2010). The second method of seeding EPC is to use peptides that can enhance endothelial cell proliferation and adhesion. But the peptides don't specifically affect a single cell type.Velevaand others(2008)used a combinatorial peptide library and a phage display technique to isolate peptides to form active peptide sequences that can bind terpolymers and generate a stent surface suitable for endothelial cell adhesion. The third method of capturing EPCs is with magnets on the intravascular stent surface. This approach requiresin vitroculture and labeling of EPCs because it is nearly impossible to capture EPCs instantly without magnetic labeling of circulating blood beforehand. The final method is to screen and use EPC-targeted aptamers to specifically capture EPCs from the bloodstream. Aptamers are a class of nucleic acid moieties that have the ability to bind to a wide variety of target molecules with high affinity and specificity, such as peptides,proteins, drugs, organic and inorganic molecules and even whole cells.Hoffmannand others(2008)synthesized for the first time an EPC-specific aptamer that exhibits high specificity for EPCs and can function rapidly to repair damaged endothelium and can inhibit intimal hyperplasia.

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Sandhya R. Panch, Harvey G. Klein, euHematology (Seventh Edition), 2018

Hematopoietic stem cell collection

Blood cell separators developed for hemapheresis are used to collect peripheral blood cells for cell therapy. The most common application of apheresis technology for cell therapy indications is the collection of HPCs from peripheral blood after administration of recombinant hematopoietic growth factor or chemotherapy, or both, to “mobilize” large numbers of HPCs into the circulation. The fraction of mononuclear cells collected contains a subset of progenitor cells that, when infused, can lodge in and reconstitute the bone marrow of patients receiving ablative radiation or chemotherapy, or both. Likewise, donor leukocytes obtained from leukapheresis procedures can be used for infusion of donor lymphocytes or subject to further treatment for immunotherapy.

Peripheral blood hematopoietic stem cells and stem cells

It has long been known that pluripotent HPCs and possibly primordial hematopoietic stem cells capable of reconstituting the bone marrow and immune system circulate in peripheral blood. Numerous studies have confirmed the potential for rapid and durable engraftment of hematopoietic stem and peripheral blood progenitor cells (PB HSPCs), mobilized in the circulation by hematopoietic growth factors harvested by high-volume leukapheresis procedures and subsequently administered to patients with myeloplasia after discharge. chemotherapy doses. The concentration of hematopoietic stem cells and progenitor cells in the peripheral blood can be increased by administering cytokines such as granulocyte colony stimulating factor (G-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF) and by administering chemotherapy . CD34+Peripheral blood hematopoietic cell counts generally increase 20 to 40 times (from 1-3 cells/µL to 40-70 cells/µL) after administration of G-CSF alone and increase 100 to 1000 times with chemotherapy The rebound is increased by administration of G-CSF. Efforts to define optimal collection conditions have been limited by the lack of a standardized assay; however, the time and level of increase in leukocytes and CD34 in the donor's peripheral blood+cell counts after administration of mobilizing agents have been shown to be useful indicators. Despite continuing problems with interlaboratory standardization, flow cytometric analysis of cells labeled with a fluorochrome conjugateCD34 antibodyused for making "real-time" decisions about the timing and appropriateness of PB HSPC collections. Several prestimulation donor variables are important. Increasing age, white ethnicity, and female gender are associated with significantly lower post-G-CSF CD34+cell count, which would favor younger men as donors when high doses of cells are needed.26

Various clinical scale devices for CD34+cellular enrichment of leukapheresis pools by immunosorbent or immunomagnetic techniques are available. This "positive selection" of CD34+hematopoietic cells also result in reduced numbers of cells that do not express the CD34 antigen, such as T lymphocytes. T cell reduction products may provide a lower risk of GVHD and the ability to produce CD34-enriched apheresis products+cells facilitate graft manipulations for experimental cell therapies such as ex vivo CD34 expansion+cells. However, some studies suggest a higher incidence of graft failure and possibly delayed immune reconstitution in certain patient populations when transplants are performed with highly purified CD34-selected T cell-depleted products.

In the last decade, PB HSPCs have overtaken bone marrow HSPCs as the predominant graft source in the clinical setting for various hematopoietic stem cell transplants. PB HSPCs are the graft of choice in 99% of conditions where autologous transplants are indicated. For allogeneic transplants, especially those using stem cells from matched unrelated donors or in pediatric transplants, bone marrow stem cells are still the popular graft source. PB HSPC transplants result in higher rates of clinically significant chronic GVHD in these situations.Table 118.3provides an overview of comparisons of bone marrow versus PB HSPCs in various types of allogeneic stem cell transplants.

From the donor's point of view, the discomfort and inconvenience of several days of cytokine administration can be a significant impediment. Most donors experience some degree of bone discomfort and pain, and some donors require hospitalization due to more serious side effects from G-CSF administration. However, a large prospective study by the National Marrow Donor Program found that the intensity and duration of pain in the peri-harvest period (during G-CSF administration before apheresis between PB HSPC donors and after the bone marrow biopsy procedure in HSPC bone marrow donors) was similar for individuals undergoing HSPC collection by apheresis or bone marrow biopsy. Furthermore, 3% of bone marrow donors reported ongoing low-grade pain at the site 6 months after completing the procedure, whereas recovery was complete for PB HSPC donors at this time. Risk factors for worse toxicities, including older age, female sex, and obesity, were similar between bone marrow and PB HSPC donors.27While concerns remain regarding the hypothetical long-term effects of exposing healthy donors to growth factors, another recent prospective study with more than 20,000 donor-years of follow-up showed no evidence of increased risk of cancer, autoimmune disease, and stroke in donors receiving G - CSF.28Enlargement of the spleen commonly occurs, and several cases of splenic rupture have been reported. Citrate toxicity is a common complication of PB-HSPC collections; Although usually mild and transient, it can be serious and even fatal in some people (see previous section). Venous access requires large-bore multilumen catheters, and these appear to be particularly susceptible to clotting, especially when patients receive recombinant cytokine stimulation. Bleeding, particularly in thrombocytopenic patients, is another potentially serious complication of central venous catheter placement.

Some patients who have received several previous cycles of chemotherapy and a small proportion of normal healthy donors, called "mobilizer few", do not respond adequately to mobilization regimens. In these circumstances, Plerixafor, a reversible CXCR4 antagonist that can block adhesion of HPCs to bone marrow stroma, may enhance aggregations by releasing these cells into the circulation. In a randomized controlled trial, the combination of G-CSF and Plerixafor resulted in a significantly greater proportion of patients with non-Hodgkin's lymphoma reaching their target dose of cells in fewer days of apheresis.29Similar success is achieved with multiple myeloma patients who achieve a 4.8-fold increase in peripheral blood CD34 cell count compared to 1.7-fold with G-CSF alone. New evidence indicates that Plerixafor can be administered "just-in-time" to rescue collections for patients who mobilize poorly on G-CSF alone. Although the ideal circumstances and timing of administration have yet to be determined, some guidelines recommend adding Plerixafor to circulating CD34+cell count less than 20 × 106/L on two consecutive days accompanied by an increase in leukocytes.30

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Microfluidic devices for stem cell analysis

D.-K. Kang, ... W. Zhao, emMicrofluidic devices for biomedical applications, 2013

(Video) Humanized M NSG PBMC and CD34+ Mouse Models 2023 | Applied StemCell, Inc.

11.6 Microfluidics-printingceleseparationsteknologi

The detection, isolation and classification of specific stem cell subpopulations are important both in basic research and in the clinical applications of stem cell-based therapies. Advances in microfluidic cell sorting have allowed researchers to achieve improved separation with relative ease and considerable time savings.Abandonedand others, 2009; Kermer, 2005). Based on the detection method used, microfluidic cell sorting devices can be classified as either label-dependent or label-free (Figures 11.22–11.26). The marker-dependent approach is based on identifying specific expression levels of cell surface proteins. For example, thermoresponsive microfluidics has been used to selectively release captured blood cells (Fig.11.22) (or cucumberand others, 2011). In this case, the authors used anti-CD4 and anti-CD34 antibodies immobilized in a microfluidic device to capture CD4+ and CD34+ cells from the blood. Cooling the microchip below 32°C led to the release of the collected cells. 195 CD4+ cells and 19 CD34+ cells per million blood cells were quantified without labeling using this affinity microfluidics device. Optical switching-based fluorescence-activated microfluidic cell sorters have also been developed to isolate cells that express green fluorescent protein (GFP).Fig.11.23) (Wangand others, 2004). When cells expressing GFP are detected and determined to be a target cell, the optical switch is activated and a focused laser spot diverts the cell to the target output channel. The laser dot is translated at a speed appropriate to the flow rate and at a small angle to the flow axis to maximize the interaction time between the laser and the cell. The resulting lateral displacement of the cell along the flow stream is sufficient to ensure that it is directed towards the target outlet channel. Additionally, MACS has been achieved in microfluidics for stem cell extraction (Fig.11.24) (tannedand others, 2005). Magnetic beads conjugated with anti-CD31 monoclonal antibody (PECAM1) were introduced into the microfluidic device to remove human umbilical vein endothelial cells (HUVEC) from a mixture of HUVEC and hMSC cells introduced into the device at the same time through the second channel. The two streams are thoroughly mixed in the micromixer and the cell-bead complexes are separated in the buffer liquid by an external magnetic field. For the unlabeled approach, unique biophysical characterizations such as membrane size and electrophysiological properties are needed to allow separation of unlabeled cells in microfluidic devices. Various microfluidics-based cell sorting devices used to isolate stem cells are summarized inTable 11.1.

CD34 Antibody - an overview (1)

11.22.Microfluidic cell sorting devices based on marker-dependent or label-free approaches. Thermo-responsive microfluidic chip designed to release selectively trapped blood cells.

(Source: Reprinted with permission from Gurkanand others, 2011.) (a) The microfluidic chip is composed of three parallel channels (4mm×22mm×80mm), one of which (middle channel) is used as a temperature indicator channel. Blood is introduced into the upper and lower delivery channels by manual pipetting. (b) The central channel is coated with a temperature-sensitive liquid crystal dye, which responds to temperatures between 35°C and 40°C. The target temperature is maintained in the middle channel. (c-f) Scheme of the operating principle of unmarked selective capture of whole blood and controlled release of cells in thermoresponsive microfluidic channels.

CD34 Antibody - an overview (2)

11.23.Layout of a microfluidic cell sorting system showing sorting junction and optical switch.

(Source: Reprinted with permission from Wangand others, 2005.) Cells in the sample are aligned to the center of the channel by flow focus. Fluorescently labeled target cells are analyzed and detected by turning on the optical switch. Target cells are directed by the laser to the collection outlet, while all other cells flow to the waste outlet.

CD34 Antibody - an overview (3)

11.24.Micromagnetic separators for stem cell sorting.

(Source: Reprinted with permission fromtannedand others, 2005.) (a) Lamination micromixer with 180° rotation. (b) Geometry of a single mixing unit. (c) Prototypem- IMCS.

CD34 Antibody - an overview (4)

11h25.DEP-based classification of microfluidic cells.

(Source: Reprinted with permission fromPrietoand others, 2012.) (a) PDMS glass microfluidic device for capturing DEP. (b) The device has three capture sites with castellated electrodes that can be addressed independently. The initial cell mixture flows through all three capture sites. (c) Collection is performed with three perpendicular flows after isolating each capture zone by closing adjacent pneumatic valves.

CD34 Antibody - an overview (5)

11.26.Microfluidic device for separating AF MSCs using lamellar array structures.

(Source: Reprinted with permission from Wuand others, 2009.) (a) Schematic illustration of the cell sorting chip. (b) Detailed view showing which spheres or cells can be focused to form a narrow stream. (c) and (d) Detailed dimensions of the chip and cell separation mechanism. (c) The larger spheres or cells are separated by the lamella-like structures. (d) The smaller beads or cells flow through the space between the lamella-like structures.

Table 11.1.Microfluidic devices for isolation/separation of stem cells

isolation methodinsulation detailssegmented cellsTransport medium and control cells% recovery% purity/fold increasecursor dependentLa bel-friReferences
cell affinityImmobilized microfluidic channel of anti-EPCs marker antibody (CD23, VEGFR-2, CD31 and CD146)EPCsMSCs, VSMCs, VECsN / DN / DEEPlouffeand others, 2009; or cucumberand others, 2011b
cell affinityPNIPAAm microfluidic channel immobilized with CD34 antibodyEPCsHelblodN / D19 cells/106blood cells (EPCs/WBCs + RBCs)EELiuetal., 2005
magnetic microsphereCD31 antibody-conjugated magnetic beadCD31 hMSCs (hMSCs)HUVEC90,2N / DEEWalking stickand others, 2005a
magnetic microsphereMagnetic bead conjugated with anti-SSEA1 antibody.SSEA-1+mESC'erheterogeneous mESCs (SSEA-1+mESCs and SSEA-1mESCs)N / D95~99,5EEsousaand others, 2011
DEP based microfluidicsN / DCD34+ hematopoietic stem cellsCollection of stem cells from bone marrow and peripheral bloodN / D5 times enrichmentEEtallyand others, 1995; Stephense a1., 1996
DEP based microfluidicsN / DNG2+ cells AND Nestin+ cells (presumed progenitor cells)Fraction of nucleated cells isolated from fat
tissue and most erythrocytes
N / D(1.9% x 28%) / 14 times increaseEEhe looked outand others, 2008b
DEP based microfluidicsN / DneuronsNeurons and NSPCs,N / D1.4-fold enrichment for neuronal cultureEEPrietoand others, 2012
Sizelamellar matrix structureAFMSCsEndothelial cells in amniotic fluid97,1%N / DNoEWuand others, 2009a
SizeMicrofluidics integrated into the porous membraneHSCsbone marrow98%N / DNoESchirhagland others, 2011

11.6.1 Cursor dependent approach

Fluorescence activated stem cell sorting

FACS, the gold standard for conventional stem cell sorting, has also been developed in microfluidics to isolate rare cells. Although the development of a practical technology for FACS based on microfluids has proved challenging, Wangand otherstook a unique approach to developing this technology by first integrating an optical coupling system for fast (2 to 4 ms) active control of cell routing into a microfluidic chip (Fig.11.23) (Wangand others, 2004). Using all-optical switching, a fluorescence-activated microfluidic cell sorting was implemented and evaluated. This study suggests the possibility of integrating FACS into a microfluidic chip to allow for more accurate real-time stem cell analysis.

Selection of magnetically activated stem cells

MACS, one of the most popular conventional cell isolation methods, has recently been developed in microfluidics to isolate rare cells. Tan and his colleagues first introduced micromagnetic sorters for stem cell sorting (Fig.11.24) (tannedand others, 2005). A 3D mixer was integrated into a microfluidic channel to achieve lamination with 180 degree rotations and rapid mixing between cells and magnetic beads. To isolate the target cell from the mixture, magnetic beads conjugated with CD31 antibodies were used to remove CD31+ endothelial cells with an external magnetic field. Up to 90.2% of hMSCs were isolated and recovered. Furthermore, Souse and his colleagues introduced a two-input/two-output microfluidic device to isolate mouse mESCs using superparamagnetic particles. To isolate embryonic specific antigen 1 positive mESCs (SSEA-1+) from a heterogeneous population of mESCs, anti-SSEA-1 antibodies were conjugated to superparamagnetic beads and mixed with the cell mixture. Once the mixture was injected into the microfluidic channel and the magnetic field was applied, the SSEA-1+ mESCs deviated from the laminar flow direction according to their magnetic sensitivity and were thus separated from the SSEA-1mESCs.

Stem cell isolation based on cell affinity

Affinity chromatography is one of the most popular methods for the separation, isolation and purification of target biomolecules from whole mixtures based on highly sensitive and specific interactions between antigen and antibody, or receptor and ligand. Brian and his colleagues have developed microfluidics-based cell affinity devices that have the ability to capture circulating endothelial progenitor cells (EPC) (Plouffeand others, 2009), which suggests thatpotential application for MSC isolation. Gurkan and his colleagues developed a thermoresponsive microfluidic device to selectively release captured blood cells (Fig.11.22) (or cucumberand others, 2011). Human anti-CD34+ antibodies were immobilized in a PNIPAAm microfluidic channel at 37°C; when blood containing CD34+ stem cells was injected into the canal (eg, to capture CD34+ EPCs), CD34+ cells were successfully captured from whole blood. To release the captured cells, the microfluidic device was cooled below 32°C. Released cells showed greater than 90% viability in a live/dead assay.

11.6.2 Approach without label

Electrophysiological properties

Isolation of specific progeny and progenitor cells based on their distinct behavior during exposure to an alternating electric current field represents a new approach in the field of stem cells. Unlike conventional techniques, which rely on the presence of specific cell surface markers, DEP separates cells based on the intrinsic dielectric properties of the cells without the need for labeling. Talary and colleagues first demonstrated the potential use of DEP in an HSC classifier (tallyand others, 1995). Applying a sinusoidal alternating current field (6V peak-peak at 5kHz), they found a 5.9-fold enrichment in the CD34+ cells (0.84-4.97%) captured on the integrated castellated electrode. Similarly, Stephens and colleagues used the same platform to isolate CD34+ cells directly from diluted peripheral blood stem cells. They demonstrated an almost five-fold increase in the frequency of CD34+ cell populations in the fractions collected in the 5-10 kHz range. They further confirmed that the isolated CD34+ cells were able to form colonies using a colony formation assay. These two studies demonstrate the possibility of using DEP as a non-invasive, label-free screening approach in the stem cell field.

Recently, Prieto and his colleagues developed a dielectrophoretic-assisted cell sorting matrix (DACS), which consists of an array of DEP electrodes with three multiplexed capture regions that can be triggered independently at different frequencies. This device was used to separate a mixture of NSPCs and differentiated neurons (Fig.11.25) (Prietoand others, 2012). They reported the first statistically significant neural cell sorting using DACS to enrich neurons from a heterogeneous population of mouse-derived NSPCs and differentiated neurons. A 1.4-fold neuronal enrichment was obtained. Furthermore, Vykoukal and co-workers have developed a dielectrophoretic field current fractionating separator (DEP-FFF) using a novel microfluidics-microelectronics hybrid flexible circuit fabrication method. They used DEP-FFF to separate putative stem cells (NG2+) from adipose tissue (he looked outand others, 2008). In this study, a cell fraction with nuclei of cell debris and the majority of the erythrocyte population was isolated from adipose tissue. By introducing the cell fraction into DEP-FFF, they found a 14-fold enrichment in the NG2+ cell population (2-28% purity). They further identified that the enrichment of NG2+ cells is coincident with the enrichment of Nestin+ cells, indicating that cell isolation occurred at an early stage of development. These studies imply that the intrinsic dielectric properties of cells can be used as a label-free approach to isolating stem cells for transplantation.

size differences

The difference in size between different types of cells and between subpopulations of HSCs has become well known and can be used as a parameter for stem cell classification. For example, the presence of MSCs in amniotic fluid (AF) is an attractive source of cells, as MSCs do not have the same ethical issues that surround ESCs. A variety of cells are present in AF, including stem cells, amniotic fluid cells, and many dead epithelial cells with diameters of 4-6μm, 15-20μm, and 40-60μm, respectively. With this in mind, Wu and his colleagues developed a microfluidic device to isolate MSCs from amniotic fluid using a combination of a T-junction focus structure and a lamellar-like structure to isolate MSCs from amniotic fluid (AFMSCs) (Fig.11.26) (Wuand others, 2009a). Another example of size-based stem cell isolation was recently introduced by Schirhagl and colleagues (Schirhagland others, 2011). Its layered microfluidics device is engineered to generate a sorting device that can vent and filter simultaneously. The top and bottom layers contained channels and pressure valves, while the middle layer was a porous PDMS membrane (pore size: 10, 15 or 20μm). The porous membrane was fabricated using soft lithography and was embedded between the microfluidic layers. As HSCs are larger in size than other cells present in bone marrow, with the exception of monocytes, HSCs have been successfully isolated from human bone marrow samples without any sample pretreatment. At smaller pore sizes (≤15μm), the efficiency of stem cell isolation increased significantly.

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Dose determination for stem cell drugs

James L. Sherley, Iperinatal stem cells, 2018

Pseudodosing, the practical answer to the problem of distributed stem cell enumeration

Despite the lack of means to quantify the dose of DSC, stem cell medicine has not progressed without a dosing baseline. Consistent with the general importance of dose in medical principles and practices as described above, researchers and clinicians have developed surrogate dosing metrics that account for the correct principle desired, which is the specific dose of DSC. As illustrated in the following discussion, it would be more appropriate to refer to these surrogates aspseudodosagembecause the essential principles of treatment or research, DSCs, do not actually count in determining the dose.

In the case of HSC transplant therapies, the most important pseudodosing metric is CD34+phenotype[3-10]. HSC transplantation is the only stem cell therapy approved in routine clinical practice and as such is also the stem cell therapy with the longest and most extensive clinical experience. A portion of HSC transplant treatment preparations are stained with fluorescentanti-CD34 antibodies are conjugated or detected by fluorescence and positive cells are scored by epifluorescence microscopy or flow cytometry. As mentioned earlier, the vast majority of cells detected with this biomarker are CPCs, not HSCs. Despite this limitation to determine the absolute count of CHS, CD34+cell count has been shown to be a clinically useful predictor of clinical outcomes in many HSC transplantation scenarios[3-6].

Several studies have shown dose-response relationships between CD34+cell number and HSC engraftment success. Importantly, these studies focused on establishing a universal critical level of CD34 administered+hematopoietic cells necessary to ensure a high probability of effective restoration of hematopoiesis[3-6]. But over time it became clear that the differences in CD34+cells from different sources do not always reliably predict clinical outcome[11]. This problem has become more evident in evaluations of HSC transplant therapies, where two independent sources of cord blood have been combined to obtain sufficient CD34 numbers.+cells to ensure therapeutic engraftment. In this configuration, the number of CD34+cells in the respective cord blood samples were not predictive of which progeny cell from the source would predominate in the recipient patient[8]. Given the parent-child relationship between HSCs and hematopoietic CPCs, the existence of a consistent relationship was a plausible hypothesis, but would likely not prove correct with further investigation. In addition to disruptions in the relationship due to cell isolation procedures, many physiological factors affect the rate at which HSCs divide to produce committed hematopoietic cells or for self-renewal.[12], ensuring unpredictable inter-individual and constitutional variability in the relationship[11]. These factors may not be apparent when an optimal or saturated level of HSCs is administered. However, with pooled cord blood samples, although the total number of HSCs may be sufficient for successful engraftment, individual populations remain determining the rate of their own persistence. The results obtained in this limiting condition may manifest the reality that, although CD34+cells are certainly related in some quantitative way to HSCs, their proportion is not directly proportional[11].

Other commonly transplanted types of stem cells - primarily mesenchymal stem cells isolated from a variety of tissues[13]— lack of unique markers with the degree of stem/progenitor specificity shown by hematopoietic cell biomarkers. Combinations of surface biomarkers are often used to characterize their preparations, with no specificity sufficient to quantify the stem cell fraction of these very heterogeneous cell populations. In these cases, the total nucleated cell count (TNC) and/or the related mononuclear cell count (MNC) are used as a pseudo-dose metric to relate differences in dose. TNC is also often used to complement CD34+cell fraction for HSC transplantation therapies. In contrast to CD34+phenotype, its relationship with the DSC fraction is unreliable[3,4,8]. Although widely used as meaningful quantitative measurements of DSCs, none of these measurements provide an effective means of determining the absolute dose of the crucial principles of stem cell transplant medicine, which are therapeutic DSCs.[11](Fig.28.4).

CD34 Antibody - an overview (6)

Figure 28.4. Conceptualizing distributed stem cell (DSC) pseudo-dosing.

The diagram illustrates two common forms of pseudo-dosing for DSCs. Total Nucleated Cells (TNC) counts all intact nucleated cells in a treatment sample. Includes relatively rare DSCs (yellow diamond room), lineage-committed progenitor cells (CPCs;blue room) and committed differentiated cells (green space). Many external and constitutional factors intervene to prevent TNC dose from having a consistent relationship with DSC dose. Depending on the relative proportions of CPCs and committed differentiated cells, the TNC dose can provide a consistent indication of the CPC dose (dotted lineand proportional dimensions ofblue room). In contrast to the dose of TNC, CD34+The cellular CPC dose can serve as a semi-quantitative surrogate for the DSC dose.

Given that pseudodosing is so prevalent in the current practice of stem cell medicine, it is instructive to consider its impact on the quality of stem cell medicine and biomedical stem cell research.[11]. It is possible to assess internally consistent dose-response relationships based on CD34+and the TNC counts if enough source material is available. Sufficient cells can be obtained by pooling tissue cell samples from multiple donors. However, these research strategies are usually not feasible because the tissue of origin is often sparse and only sufficient to treat a single recipient. This usual situation is further limited by the well-known difficulty of expanding many types of potentially therapeutic DSCs by in vitro cell culture.[14]. Therefore, clinical trials are not conducted with evaluated patients who receive transplanted cells from a common cell pool. In fact, many clinical trials are based on autologous cell transplantation, so that each study patient receives a different stem cell preparation.[13]. In the case of allogeneic transplant-based trials, the challenges of identifying a sufficient number of compatible donors result in a similar situation of cell limitation. Therefore, in the typical stem cell transplantation clinical trial today, it is completely undetermined how the amount of the crucial principle tested - transferred DSCs - relates to any clinical outcome.

Since the crucial denominator for all clinical trial comparisons, which is the change in the effective dose of DSC, is not measured in pseudo-dosed clinical studies, the observed variation in clinical outcomes cannot be related to the properties of DSCs. Even in case every patient benefits greatly from a cell transplantation treatment, the cause may not be the DSCs that should be present in the transplanted specimens. Formally, other cell types in complex populations of transplanted cells or even non-cellular factors may be responsible.[11]. This misattribution may be recognized if variation in DSC scores has been poorly correlated with clinical outcome, but pseudodosing cannot provide such discrimination. Pseudodosing confounds any clinical trial analysis in this way, including comparisons of different patients, different numbers of transplanted cells, different treatment regimens, different clinical trials, and so on.

Pseudodosing excludes other important properties that promote clinical trials and higher quality medical care. If it were possible to monitor DSC number and function specifically, it would be possible to detect agents and conditions that alter DSC viability and function for beneficial or deleterious effects. For example, the author suggested that some immunosuppressive agents used for DSC allogeneic transplantation therapies may reduce DSC self-renewal, thereby compromising DSC engraftment rate[15]. The ability to monitor the effects of reagents and treatments on DSCs in vitro could be particularly advantageous for gene therapy and gene-edited stem cell medicine. For lifelong cures, both gene therapy and gene editing manipulations must occur in long-lasting regenerative DSCs[16]. However, if the necessary genetic modifications are not effective, or if they damage targeted therapeutic DSCs, the effectiveness of these treatments can suffer dramatically. The ability to monitor targeted DSCs during these procedures would allow for optimization and ensure a sufficient number of modified DSCs transplanted for more effective therapies.

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Embryonic toxic lesions and stem cell therapy

Vadim B. Popov, ... Lilia V. Shabasheva, euReproductive and developmental toxicology (second edition), 2017

gem sack

At E 7, the presumptive YS mesodermal cells aggregate with each other. Cells at the central focus of these aggregates transform into hematopoietic cells, and the peripheral population flattens and differentiates into endothelial cells to form blood islands.McGrath et al., 2003; Boucher and Pedersen, 1996; Zone, 1995; Palis and others, 1995). These cells are proposed to have a common progenitor, the hemangioblast (McGrath et al., 2003; Shalaby et al., 1997; Choi e outros, 1998). Proof of this concept is provided by the combined expression of some genes in hematopoietic and endothelial cell lines. In particular, a vascular endothelial growth factor (VEGF) has been shown to form blast colony-forming populations of cultured cells that form populations of endothelial and erythroid cells via embryoid bodies (EBs) (Choi et al., 1998; Millauer et al., 1993; Kallianpur et al., 1994; Watt et al., 1995; Eichmann et al., 1997; Kabrun et al., 1997; Kennedy and others, 1997). The most convincing evidence for the existence of hemangioblasts comes from research results on the transformation of EB colonies formed from mouse embryonic stem cells (mESCs). Hematopoietic development in EB cell differentiation forms macrophages as well as erythroids and mast cells. Mouse embryos do not have many specialized cells in the early stages of hematopoiesis, but their progenitors are still present in the general hematopoietic cell population and actively participate in the formation of definitive lineages, specifically erythroblasts, macrophages and mast cells, which then migrate to other hematopoietic tissues, the liver. Macrophage progenitors appear simultaneously with primary erythroid cells and their population increases sharply at E 8.25–9.5 (from 2–6 to 26–29 pairs of somites), after which a decrease in population is observed. Mast cell progenitors appear a little later (E 8.6; 9-16 pairs of somites) (Palis and others, 1999), and they usually mature elsewhere rather than the YS pathway (Lacaud and others, 2001). The use of CD34 antibodies allows revealing the common and still hypothetical cell - the hemangioblast, which provides a much clearer view of early vasculogenesis, hematopoiesis and progenitor cell production, and allows tracking the fate of cells in formation. In addition, the possibility of analyzing mutations in the place of VEGF, responsible for the normal development of embryonic blood vessels, or in the two VEGF receptors (FLT1 or Flk1), also serves to better understand angiogenic processes (Breier and others, 1992).

The fact that primary hematopoietic cells develop in the YS gives the impression that this organ is the source of all hematopoietic populations in the developing organism (ie, it is the source of HSCs). However, mouse embryonic hematopoiesis occurs not only in the YS, but is also consecutively activated in various organs. The taller stem is characteristic of definitive erythroblasts, which, like mast cells, are present in the YS loop but are unlikely to mature there. The entire population is produced for export to the liver (Palis and others, 1999). At the same time, experimental evidence suggests that the major source of all hematopoietic progenitors is the embryonic region that forms the aorta, gonads, and mesonephros (AGM region), specifically the para-aortic splanchnopleural region.Godin et al., 1995; Muller et al., 1994). To find out which embryonic region is the source of HSCs,Müller et al. (1994)transplanted tissues from these regions into irradiated adult recipient mice to show that cells from the para-aortic splanchopleural region were the only cells thatform a long-lived multilineage population of HSCs that exhibited activity before YS and hematopoietic liver cells. Analysis of hematopoietic tissues (blood, thymus, spleen and bone marrow) revealed В and Т lymphoid cells, macrophages and mast cells.

Cells generated in YS can be progenitors of liver and bone marrow blood cells. Progenitor hematopoietic cells populate the liver at the end of E 9. ThusYoder et al. (1997)transplanted E 9.0 YS cells (c-kit+/CD34+) into the liver of newborn animals treated with busulfan and obtained evidence of repopulation of these cells throughout life. Such findings raise new questions, specifically what is the role of primitive YS erythroblasts, whether the latter contain progenitor cells, whether the hemangioblast exists and what function it performs. The answers to these questions can only be found during YS formation: before and during blood islet formation. At E 8, several primitive red blood cells, erythroblasts, form on these blood islands and actively divide and mature there. The blood islands fuse to form the primary capillary network (E 8.5). The nuclei of primitive erythroid cells are quite large; they maintain this size throughout the life cycle and produce the embryonic form of hemoglobin. Single cell macrophages have also been detected in blood smears (Brotherton and others, 1979). After the capillaries intertwine, vessels of various sizes develop, and especially after the paired endocardial heart tubes merge into a single heart tube and the heartbeat begins (E 7.0–8.0), the vascular system acquires a hierarchical structure . Part of the vessels are located extraembryonic in the visceral wall of the YS (allantois), and the rest are formed by angioblasts that migrate to the embryo to form endothelial filaments along the embryonic axial structures and later differentiate into the dorsal aorta, cardiac veins, etc. . Thus, the embryonic/extraembryonic circulatory systems include vascular, hematopoietic, and cardiac components, each developing from specific mesodermal regions (McGrath and others, 2003).

Primary hematopoiesis and vasculogenesis are associated with the inner tissue of the YS visceral wall (splanchnopleura). The splanchnopleura of rats and mice is twofold: the outer layer, endoderm, provides transport, metabolism, and synthesis of maternal macromolecules, and the inner layer, mesoderm, produces primitive blood cells. In recent years, evidence has shown that the visceral endoderm is an important source of regulatory signals for the formation of blood cells and endothelial networks.Nath et al., 2004.). In particular, NO is considered a vasoactive agent. Nitric oxide fulfills many functions in mammalian embryogenesis and acts as a signaling molecule and free radical.Natal., 2004). It is suggested to play an active role in parthenoocyte maturation, preimplantation embryo preparation, and regulation of gene expression.Gagioti et al., 2000; Bogdan, 2001). Inhibition of NO production in pre-implantation mouse embryos inhibits development and reduces embryonic survival rates.Biswas et al., 1998). YS visceral endoderm also produces NO, and inhibition of NO production ends blood island formation, vasculopathies, and stops embryonic development.Nath et al., 2004.). Impaired development of visceral YS endoderm inhibits vasculogenesis and erythropoiesis initiated in embryoid bodies.Bielinska and others, 1996). Ephrins, Tie2, angiopoietin, platelet/endothelial cell adhesion molecule, VEGF (Flk1) and other molecules are also essential for mammalian vasculogenesis, and their loss or deficiency leads to vascular dysfunction, cardiovascular defects and embryonic lethality.Gerety and Anderson, 2002; Sato et al., 1995). In particular, mouse embryos lacking the receptor tyrosine kinase, Flk1, fail to form the extraembryonic circulatory system and die at E 9.5 (Shalaby et al., 1997).

(Video) Optimizing CD34+ Cell Genome Editing for Efficiency and HSPC Maintenance

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The evolution of coronary stents: A brief review

Trevor Simard MD, ... Edward R. O'Brien MD, emCanadian Journal of Cardiology, 2014

Endothelial stem cells

First described by Asahara and Murohara,85Endothelial progenitor cells (EPCs) are a subset of circulating cells that have been implicated in vascular homeostasis and endothelial repair,34,86-88thus increasing its profile in a wide range of vascular diseases.20,89EPCs are believed to increase vascular re-endothelialization, targeting areas of injury and differentiating into mature endothelial cells and/or influencing mature endothelial cells through paracrine signaling. Indeed, increased cardiovascular risk has been associated with reduced numbers of circulating EPCs.35and decreased EPC function were associated with the development of ISR.20,90This intrinsic repair mechanism is therefore beginning to be targeted in the development of certain new therapeutic stents.

The first attempt to apply our understanding of EPC biology to stent design was the development ofCD34 antibodycoated Genous stent. In a marked departure from its drug-eluting contemporaries, its aim was not to inhibit cell proliferation, but rather to bind circulating EPCs via its hematopoietic marker.19,91in an attempt to improve stent re-endothelialization. In its first human record, the Genous stent was implanted in 16 patients with 1 case of TVR observed in 9 months.92Subsequent studies in ST-segment elevation AMI93and high-risk patients94found acceptable safety and efficacy profiles. However, in a recent single-centre study of 193 patients comparing it to Taxus Liberté, it was associated with a non-significantly higher rate of ISR at 1 year.95The study, planned to include 1300 patients, was stopped early because of this suggestion of higher rates of target vessel failure.

The failure of the Genous stent may, in part, reflect an incomplete understanding of the biology of EPC and/or a mistaken emphasis on 'capturing' EPC. As mentioned earlier, EPC dysfunction, in addition to reduced EPC numbers, is associated with the development of ISR.20,90Improving EPC function at sites of vascular injury therefore represents a natural alternative strategy. Indeed, we report encouraging results in animal models using this alternative approach, including enhanced reendothelialization and reduced neointimal formation after stent implantation.34,87Despite this, negative results in clinical trials may not mark the end of stents designed to explore the intrinsic mechanisms of reendothelialization, given the unique advantages of this approach – particularly its possible implications on the need for platelet inhibition. It is conceivable that early reendothelialization reduces the risk of TS and thus allows early discontinuation of dual antiplatelet therapy. The GATEWAY registry, which specifically investigates early discontinuation of dual antiplatelet therapy with Genous stents, may suggest a niche for this stent in patients at high risk of bleeding.

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CD34 Antibody - an overview? ›

The CD34 antigen is a single-chain transmembrane glycoprotein, expressed on human hematopoietic stem and progenitor cells, endothelial progenitor cells, vascular endothelial cells, embryonic fibroblasts, and some cells in fetal and adult nervous tissue.

What is CD34 a marker for? ›

CD34 is routinely used to identify and isolate human hematopoietic stem/progenitor cells (HSPCs) for use clinically in bone marrow transplantation, but its function on these cells remains elusive.

What is CD34 antibody? ›

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen.

Is CD34 a tumor marker? ›

CD34 is an antigen present in hematopoietic progenitor cells and endothelial cells. Anti-CD34 antibody is a highly sensitive marker for endothelial cell differentiation and has also been studied as a marker for vascular tumors.

What is CD34 leukemia? ›

Background: The stem cell marker CD34 is expressed by leukemia blasts only for a subset of patients with acute myelogenous leukemia (AML).


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