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Vol.2, No.6, 519-527 (2010) Health doi:10.4236/health.2010.26078 Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/ Hematopoietic stem cells from peripheral blood the perspective of non-mobilized peripheral blood Vassilios Katsares*, Zissis Paparidis, Eleni Nikolaidou, Anastasia Petsa, Iliana Karvounidou, Karina-Alina Ardelean, Nikolaos Peroulis, Nikolaos Grigoriadis, John Grigoriadis Biogenea-Cellgenea Ltd, Trade Centre “Plateia”, Thessaloniki, Greece; *Corresponding Author: vkatsare@gmail.com Received 1 February 2010; revised 21 February 2010; accepted 23 February 2010. ABSTRACT The peripheral blood is a major source of he- matopoietic stem cells. Almost for two decades the peripheral blood has been mobilized, in or- der to enhance the CD34+ concentration. The isolated stem cells from the mobilized periph- eral blood are used as an alternative, or in addi- tion to bone marrow derived stem cells. In this paper, a new perspective is being discussed; the use of non-mobilized peripheral blood as an alternative source for hematopoietic progenitor cells. The number of isolated hematopoietic stem cells is evaluated using flow cytometry. The vi- ability can be evaluated using the trypan blue exclusion test, the flow cytometry or automated assays. The isolated hematopoietic stem cells could be used for ex vivo expansion either in static systems or in proper bioreactor systems, prior to cryopreservation and/or transplantation. Keywords: Non-Mobilized Peripheral Blood; Hematopoietic Stem Cells; Ex Vivo Expansion 1. INTRODUCTION Since the early 1990s, peripheral blood progenitor cells collected by apheresis have largely replaced bone mar- row as a source of hematopoietic stem cells for autolo- gous transplantation [1]. Peripheral blood cells produce more rapid hematopoietic recovery, thereby leading to reduced costs [2-5]. Furthermore, although follow-up is more limited in the PBSC group than in the BM group, no evidence was found that the use of PBSC was associ- ated with an increased risk of chronic GVHD compared to results with BM [6]. 2. CHARACTERIZATION OF HEMATOPOIETIC STEM CELLS Hematopoietic stem cells are normally found in very limited numbers in the peripheral circulation (less than 0.1% of all nucleated cells). It is logical that progenitor cells circulate in the periphery, as this ensures an even distribution of hematopoiesis within the BM [7]. CD34 antigen expression is used as a surrogate marker for he- matopoietic stem cells and enumeration of CD34+ cells has been used to quantify progenitor and stem cell con- tent [8]. PBSCs represent a subpopulation of all CD34+ cells (CD34+/CD38–) found in the circulation [9]. CD34+ cell viability was measured by an established flow cytomet- ric method [10]. The method is based on triple staining with anti-CD34-PE, anti-CD45-FITC, and the viability marker 7-actinomycin D, and it allows the calculation of the absolute numbers of viable CD34+ cells. Recently, a new rapid and accurate method has been developed for the viability evaluation based on luminometry [11]. 3. CRYOPRESERVATION AND THAWING OF PBPCS PBPCs are usually harvested and stored in liquid N2 un- til reinfusion. Storage at this low temperature will block all enzymatic pathways and metabolism in the cell [12]. Cryopreservative(s) must be added to the PBSC before freezing in order to protect the cells. The concentration of the cryoprotectant and the rate at which the cells are frozen are the main factors governing the survival of the cells. Thereafter the cells are stored in liquid N2 [13]. Due to their different cell membrane composition and higher osmotic inactive volume, CD34+ cells are better protected from hypertonic shock and ice crystal forma- tion and should be more resistant to cryopreservation damage than the remaining nucleated cell population [14]. A computer-controlled freezer is used for the cryo- preservation. In order to ensure rate-controlled freezing an optimized program is developed and adjusted ac- cordingly. In controlled rate freezing, the concentrated stem cells are frozen down at a rate of 1-20C/min up to a tempera- V. Katsares et al. / HEALTH 2 (2010) 519-527 Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/ 520 ture point of about –40˚C. Then, the freezing process down to a target of –120˚C is performed at a faster pace, about 3-5˚C/min. For PBSCs the controlled rate freezing process is considered standard [15,16], and was in dif- ferent reports found to be superior to uncontrolled freezing approaches. This procedure is time consuming and requires staff with a specific expertise. Hence, the use of uncontrolled rate freezing in which the specimen is first cooled down to –4˚C and then directly deposited into a freezer at –80˚C or put into liquid phase nitrogen has been evaluated. Several reports [17-19] established that the uncontrolled method is safe and reveals compa- rable results to the controlled rate process for PBSCs. A controlled study performed by Perez-Oteyza et al. [20] showed that the controlled and uncontrolled rate freezing approach are comparable in terms of viability testing and that only a statistically significant decrease in the CFU- GM clonality assay could be detected in the uncontrolled freezing situation. Recent studies suggested that uncon- trolled freezing is also a viable approach for UCB stem cells [21,22]. As a cryoprotectant, a solution containing 50% DMSO in HAES-steril® 10% is used. Prior to freezing, a part of pre-cooled cryosolution (with 50% DMSO) is mixed with three parts of the buffy coat (pre-cooled), to achieve DMSO concentration of 5 or 10% in the final solution. Cryopreservation is then carried out in aliquots in cryo- genic vials. Current protocols for cryopreservation of PBPCs are usually based on the use of 10 percent DMSO in the freezing medium [23]. HPCs can be preserved with 5% DMSO, and such autografts can be safely used for stem cell rescue even after long-term nitrogen storage [24]. Several techniques for the thawing procedure have been proposed. The standard method is warming in a water bath at 37˚C until all ice crystals disappear [19]. A German study compared the thawing of cryopreserved units in a warm water bath with dry heat applied by gel pads at 37˚C. The viability and clonogenic potential were comparable, with a trend towards less infectious contamination in the dry method [25]. Different studies examined the preservation of function when thawed units were incubated at 0-37˚C [19,26]. Akkök et al. [25] suggest that even simple single- wash DMSO depletion causes significant CD34+ cell loss. Despite a beneficial impact on the frequency of adverse effects during and after stem cell infusion, this time-consuming procedure caused a delayed PLT recov- ery and increased requirement for PLT transfusions. The CD34+ cell loss, however, was never critically low, that is, never lower than 2 × 106 per kg. They concluded that single manual washing of autografts is a simple and safe procedure that decreases the frequency of adverse events during and after stem cell infusion. The procedure should be recommended especially for patients with an in- creased risk of serious toxicity, for example, patients with cardiac amyloidosis. 4. THERAPEUTIC DOSES OF PBSC Typical doses of CD34+ stem cells used for PBSCs are 2 × 106 cells/kg of recipient body weight or greater. Doses lower than this threshold is associated with pro- longed cytopenias and increased early mortality [27]. The use of higher doses of CD34+ cells may lead to quicker engraftment, particularly when doses are greatly increased [28,29]. Platelet recovery appears to be more sensitive to CD34+ doses than neutrophil recovery [29]. Efforts to enrich PBSCT by ex-vivo CD34+ cell selection (positive selection) have resulted in increased rates of GVHD, possibly by altering the cytokine expression patterns of transplanted cells or changing lymphocyte subsets delivered with the graft [30]. Autologous stem cell grafting has been used with varying degrees of success in chronic myelogenous leu- kemia (CML) [31,32], acute leukemia [33], myelodys- plasia [34], and multiple myeloma [35]. Niwa et al. [36] reported successful autologous pe- ripheral blood stem cell transplantation with a double- conditioning regimen for recurrent hepatoblastoma after liver transplantation. Nevskaya et al. [37] with preliminary results sug- gested the feasibility of therapeutic angiogenesis by lo- cal implantation of CD34+ and MNC from PB for Sys- temic Sclerosis ischemic ulcers. Improved endothelial function, stimulatory effects on circulating endothelial precursors kinetics and augmentation of microcircula- tory blood flow may contribute to therapeutic potential of the implanted cells. 5. COST ANALYSIS The cost method involved two sets of data: a data set including patient-related or direct costs, and a data set including nonpatient-related or indirect costs [38]. The patient-related costs comprise the followings: 1) hospi- talization and basic medical service, including medical and nursing staff; 2) pharmacy and blood products; 3) procedures such as operating theatre, leukapheresis and cryopreservation. On the other hand, the indirect costs comprise the clinical service department costs, for in- stance, radiology, clinical chemistry, pharmacy and the nonclinical service departments such as transportation, housekeeping and kitchen services. A study by Mishra et al. [38] reported a cost analysis using mobilized peripheral blood at 2001 prices and the V. Katsares et al. / HEALTH 2 (2010) 519-527 Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/ 521 521 costs had been recalculated into US$ by using the ex- change rates of 1st January 2001. The mean cost for the mobilization/cryopreservation phase per patient was US$ 6544 (range 5114-7273). The mean cost of high dose chemotherapy followed by hospitalization was US$ 25616 (13978-43277). This amounts to total running costs of US$ 32160 (19092-50550). Taken together, staffing, medication and blood products contributed to 74% of total costs. On average, 53% of total costs com- prised staff costs, ranging from 39 to 76%. Personnel resources varied from one center to another, from US$ 12608 to US$ 26038 per patient. Pharmacy and blood products contributed 16 and 5%, respectively, of the total costs. A study by van Agthoven [39] documented total costs of PBSC transplantation at Euro 33742. The author ap- plied a unit cost method where staff costs accounted for 42% of the transplant cost. This relatively large differ- ence in staff costs between Van Agthoven and Mishra et al. is notable, and may indicate there are cost variations between different countries, for example, related to wages. Van Agthoven [39] reported a remarkably low cost per patient for the stem cell harvesting and cryopre- servation procedures, an average of €4982, and a blood component cost (during the induction chemotherapy regimen, harvesting and transplantation phase), respec- tively, of €904, €376 and €1680, a total of €2960. In another study, Ghosh et al. [40] reported a PBSCT cost for patients with plasma cell leukemia that ranged from US$ 20000 to US$ 25000. The major part of the costs related to hospitalization, growth factors, blood products, collection and cryopreservation of PBSC. Hopefully, the use of non mobilized peripheral blood could eliminate the cost, since there are no mobilization drugs and no any special equipment required. 6. EX VIVO EXPANSION OF HSC The CD34+ surface antigen, which is a glycoprotein ex- pressed on early progenitor cells is present on less than 0.1% of the mononuclear cells in peripheral blood [41]. Many studies have shown that the minimum acceptable dose of HPCs for successful transplantation ranges be- tween 2 – 5 × 106 CD34+ cells per kg of recipient weight [42]. Furthermore, transplantation of higher doses of CD34+ cells seems to improve haematopoietic recovery and overall survival [43,44]. To try and overcome the problem of low progenitor cell dose, ex vivo expansion of CB-derived cells has been attempted. The true test of this method is whether an expansion technology will be able to provide a reliable, reproducible increase in the number of progenitor cells available from a single unit of CB, resulting in superior rates of engraftment and overall survival in adult patients. A significant hurdle of presently available methods for graft production is the ability to generate an expanded population of committed hematopoietic progenitor cells without compromising the numbers of less differentiated progenitor cells (CD34+ CD38– or CD34+ Lin– cells), which are impor- tant functional hematopoietic repopulating cells [45]. In order to consistently achieve an adequate cell dose, the processing methods must minimize cell losses. Each additional manipulation of a cellular product potentially leads to further loss of cells. In most studies, CD34+ cell selection is done before initiating cell culture [46], but the CD34+ cell selection itself is associated with a sub- stantial loss of progenitor cells. This cell loss, which may not be significant for smaller children, may become critical in reaching a suitable dose for transplant in older children and adults [47]. To achieve adequate cell doses, many researcher used different ex vivo expansion protocols, either the tradi- tional way, or using a bioreactor system. Beshlawy et al. [48] used three cytokine combinations, i.e. cell factor alone, IL-3 alone, and both stem cell factor and IL-3. Interleukin-3 enhances the amplification of early and committed progenitor cells without impairing the long-term engraftment of stem cells [49]. Several investigators reported significantly decreased cell viability after cryopreservation [50-51] and attrib- uted this to the effect of thawing and washing to remove the cryoprotectant. Laroche et al. [47] stated that thaw- ing and washing result in loss of cells approaching 20% when compared with pre-freeze counts, with the wash step responsible for nearly half of this cell loss. However, Beshlawy et al. [48], using umbilical cord blood derived hematopoietic stem cells, detected mean fold expansion of 6.64 ± 3.34 with stem cell factor alone, 7.38 ± 2.86 with both stem cell factor and IL-3, and 8.11 ± 4.49 with IL-3 alone after 2 days culture of the samples frozen for 2 weeks. There were no statistically significant differ- ences in fold expansion between the 3 cytokine combi- nations before freezing and after 1 week and 2 weeks of freezing. They concluded that although preservation procedures could decrease the count and viability of cord blood HSCs, freezing does not impair their ex vivo ex- pansion potential; however, it results in a significant loss of cell viability. In a similar study, Moezzi et al. [51] used stem cell factor, IL-3, and thrombopoietin and reported levels of expansion of (4.2-4.7 fold) after 7 days of culture of samples cryopreserved for 1 month. It was shown that a combination of early- and late- acting cytokines, including SCF, thrombopoietin (TPO), G-CSF and IL-3, resulted in only a marginal-fold expan- sion of late (CD34+) and early (CD34+CD38 –) progenitor V. Katsares et al. / HEALTH 2 (2010) 519-527 Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/ 522 cells, probably due the fact that the late-acting cytokines drive the cultures mainly toward accelerated differentia- tion [52,53]. On the other hand, cultures with only early-acting cy- tokines (SCF, TPO, IL-6 and FLT-3 ligand) resulted in better and prolonged expansion of both late and early progenitors [54], which are important for short-term early trilineage engraftment [55-57]. Peled et al. [58] suggested that TEPA supports the self renewal division cycle without compromising differen- tiation capacity of hematopoietic stem cells. A number of serum-free media have been used over the last few years with different results [59-62]. For ob- taining sufficient numbers of progenitor cells for trans- plant, FCS [63] and autologous plasma [60,61] have been used in clinical expansion protocols. However, Lam et al. [61] suggested that, with the appropriate se- rum-free media and cytokines, FCS may be excluded in clinical expansions. On the other hand, human plasma, which may contain factors that promote cell maturation [64,65] is thus unlikely to add significant value to the expansion. It has been reported that MSC constitutively secrete various hematopoietic cytokines, among them stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), leukemia-inhibiting factor (LIF), interleukin (IL)-6, IL-7, IL-8, IL-11, IL-12, IL-14, and IL-15 [66-68]. Addition of MSC as a feeder layer has been shown to improve ex- pansion of cord blood HSC, inhibit their differentiation, and decrease their rate of apoptosis [66,69-71]. Li et al. [66] demonstrated that bone marrow MSC can increase human adult PBSC expansion as compared with culture in the presence of cytokine alone. Conventional culture systems such as T-flasks and gas permeable blood bags are the most widely used devices for expanding hematopoietic cells. However, such static culture systems have several inherent limitations. Firstly, lack of mixing results in concentration gradients for dis- solved oxygen (DO), pH, cytokines and metabolites. Secondly, the environmental conditions in well-plate and T-flask are not readily monitored or controlled online. Thirdly, static systems require repeated changes of cul- ture medium, which significantly increases the risk of contamination. Hence there is an urgent need for devel- oping bioreactors for HSCs expansion, which overcomes the limitation of mass transport, keeps culture parame- ters constant and controls differentiation [72]. Several kinds of bioreactors have been applied in the field of HSCs expansion, including stirred tank bioreac- tor, fixed bed bioreactor and perfusion chamber [73-75]. It is known that hematopoietic cells are extremely sensi- tive to shear force, hence cells may suffer some physical damage under shear environment like in a stirred tank bioreactor and perfusion chambers [73]. In stirred tank bioreactor, agitation may affect cell surface marker ex- pression, including cytokine receptors, which can have a profound effect on which cells expand and to what ex- tent expansion occurs [73]. A condition with low shear but low concentration gradients is highly desirable for hematopoietic cell expansion [72]. Rotating wall vessel (RWV) bioreactor may provide a technical solution. The RWV bioreactor has several key characteristic features as follows [76]: firstly, fluid flow is near solid body and is laminar at most operating con- ditions, which avoids the large shear stresses associated with turbulent flow and allows introduction of controlled and nearly homogenous shear fields; secondly, the cul- ture medium is gently mixed by rotation, avoiding the necessity for stirring vanes, which may damage cells by both local turbulence at their surface and the high flow rates created between the vessel walls and the vanes; thirdly, there is no headspace in the RWV bioreactor while in roller bottles, due to incomplete filling of the vessel, the air in the headspace creates turbulence and secondary bubble formation in the culture medium, which are both potent sources of extra shear and turbu- lence; finally, the RWV bioreactor supports coculture efficiently by bringing different cell types of different size and density together simply and efficiently. So by optimizing the geometry of the bioreactor and opera- tional condition, it is possible to provide a uniform and low shear condition within the bioreactor. At the same time concentration gradient can be minimized. RWV bioreactors have been used to simulate micro- gravity in space flight to study how microgravity affects the hematopoiesis of astronauts [77,78], to proliferate BM cells [79]. The RWV bioreactor can provide a 3D suspension culture environment and all hematopoietic cells are suspended in the culture medium effectively, which overcomes the concentration gradients in T-flasks and makes the utilization of cytokines more effective. The National Aeronautics and Space Administration (NASA) developed two RWV bioreactors for tissue mass culture [80]. The slow turn lateral vessel (STLV) bioreactor has been used to culture several kinds of cells both on Earth and in space. The STLV was operated at 15-30 rpm on Earth and slower in space allowing a free-fall state, reducing the shear stress. The high aspect ratio vessel (HARV) bioreactor has a similar design, but the rotating speed can be slower than STLV. The NASA RWV systems have been used to study the effects of microgravity on murine HSC and evaluating the hema- topoietic homeostasis during long space expeditions [81]. The elucidation of mechanisms governing self-renewal and differentiation of HSC is needed to control the in V. Katsares et al. / HEALTH 2 (2010) 519-527 Copyright © 2010 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/ 523 523 vitro expansion. Results from pilot clinical trials of transplants using expanded UCB-HSC have shown no adverse effects in the patients. However, more clinical trials must be conducted using expanded HSC for guar- antying the safety [82]. Very recently, Delaney et al. (2010) claimed that when cord blood progenitors ex- panded ex vivo in the presence of Notch ligand we in- fused in a clinical setting after a myeloablative prepara- tive regimen for stem cell transplantation, the time to neutrophil recovery was substantially shortened. This is the first instance of rapid engraftment derived form ex vivo expanded stem/progenitor cells in humans [83]. 7. AUTHORS’ CONTRIBUTION All authors contributed substantially to this research. V.K., J.G., and N.G. designed research and collected the data; V.K., Z.P., A.P., E.N., I.K., and K.A.A. performed literature revision; V.K. analysed and interpreted data, and wrote the manuscript. All authors drafted the manu- script, revised it critically and approved it. 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Openly accessible at http://www.scirp.org/journal/HEALTH/ 527 527 GLOSSARY OF ABBREVIATIONS AND INITIALISMS BM: Bone Marrow DMSO: Dimethyl sulfoxide GVHD: Graft versus Host Disease HARV: High Aspect Ratio Vessel hESC: human Ebryonic Stem Cell HLA: Human Leukocyte Antigen HPC: Hematopoietic Progenitor Cell HSC: Hematopoietic Stem Cell LTC-IC: Long-term culture initiating colony NASA: National Aeronautics and Space Administration PBSC: Peripheral Blood Stem Cell PBSCT: Peripheral Blood Stem Cell Transplantation PPC: Primitive progenitor cell RWV: Rotating Wall Vessel STLV: Slow Turn Lateral Vessel |