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Introduction

Stem cell (SC) transplantation is very topical in the fields of developmental biology and regenerative surgery. A variety of types of SC obtained from different sources are successfully used for reparation of tissues defects. The functional rehabilitation of damaged retinas in different cases of pathology with SC transplantation is a current theme in ophthalmology [1] and can be used as a model for studying cell engrafting in brain tissue [2]. Currently we have plenty of experience in SC transplantation procedures and a huge number of published works; however it is difficult to predict the effects of such transplantation in vivo due to scarce information about SC behavior in recipient tissue. In vivo studies can’t show us all changes in SC and recipient interactions after transplantation, and it is impossible to obtain enough information about transplanted cells behavior experimentally. However, many questions about the communication of transplanted cells with recipient tissue and the processes of functional contact development and mechanisms of the new microenvironment on transplanted cells still remain open. To answer these questions new adequate model systems with the ability to investigate the destiny of transplanted cells are required. That’s why it is necessary to design ex vivo models that register individual cell behavior after transplantation and clear visualization of these changes [3]. In vitro injection of xenogeny cells into 3D rat retina culture seems to be an attractive method in this case. Additionally, organotyping explant culture damaged by laser emission is used in this work.

Materials and methods

Seven-day-old rats’ retina explant culture (DMEM/F12 with 20 ng/ml FGF and EGF, 7% FCS, В12 and N2 supplements) damaged with laser Zilos-tk (300mW, 1000 mc) was used. Neuronal stem/progenitor cells (NSPC), bone marrow stromal cells (MMSC) [4], and pigment epithelium cells (PE) of С57BL/6-Tg(ACTB-EGFP)/Osb/J GFP+ mice had been transplanted in them (300-300000 cells in 0.2mkl) in vitro. AFM data were obtained from an Atomic force microscope Solver BIO Olympus with the scanning field 100х100х7mkm3. Data analysis was done with the programs Nova (НТ-МDТ) and STATISTICA 8.0. The MMSC reaction to external irritation was estimated by fluorescent dye RH 795 after electro stimulation on an ASL-1 device.

Results

Injected SC stayed alive in culture (negative stain for propidium iodide) for more than 2 months and rapidly migrated (for longer than 3000mkm in distance) from the injection point during only the first 48 hours to the laser-damaged area. Thus injected SC had good live potency, migrated for long distances and morphologically differentiated according to a new microenvironment in all our culture systems, and these culture methods were adequate and easy-to-use 3D model systems for stem cells research.

After MMSC transplantation two populations of small (≈15mkm) rapidly migrating cells and almost motionless big (≈30mkm) cells were notable among injected MMSC. On the third day after injection into intact retinas, MMSC changed their morphology by spreading long branched outgrowths but were still negative for neural and glial cell markers (β-III-tubulin and GFAP). Analogous morphology changes in transplanted MMSC in damaged retinas were observed after 24 hours (Fig. 1). MMSC injection had a noticeable neuroprotective effect on the retina. ASM assay showed significant difference (p<0.01) between glial thickness and endothelial retina cells processes and neurites and neuro-like transplanted MMSC processes. Also it was shown that MMSC form synapses up to 2.5 ±0.06mkm in diameter on the 4th day after transplantation (Fig. 2). After electrostimulation (20V, 0.5Hz, 200ms), clear depolarization of retina neurons and their processes was detected. It was shown that some GFP+ MMSC which had changed their morphology to neuro-like after transplantation into the retina explants were able to depolarize after exogenic stimulation. This means that the MMSC population is highly heterogenic. Those cells that quickly migrated and formed neurit-like processes had an ability to generate a response to stimulation. Big (>30mkm) passive migrating cells that had taken a fibroblast-like morphology did not answer to stimulation. But it was shown that many transplanted MMSC had changed their morphology but did not respond to stimulation. Because of the absence of transplanted GFP+ MMSC and retina neuron fusion detected by fluorescent DiI staining, it is reasonable to suppose that there is a small MMSC subpopulation that has the ability for neuronal transdifferentiation.

Figure 1. MMSC morphology changes at 7 days after transplantation in retina explants. Cells positive for GFP with bipolar neuromorphology were presented (E-F). Many neurite-like processes were sprouted from MMSC during their migration and differentiation (A-D)

Figure 2. AFM analysis injected MMSC surface in full contact and semi contact mode. A, C: Processes connections between transplanted MMSC and retina cells (arrows). B: MMSC surface reconstruction after morphology changes. D: 3D neurite-like processes surface reconstruction with synaptic connections

NSPC quickly migrated (significantly differs (p<0.05) from the NSPC in undamaged explants) during the first 24 hours for a distance more than 3 mm from the damaged zone in the case when aggregates (1–5 thousands cells) were transplanted, formed neuritis and connections with the retina cells, and were detected up to 50 days in damaged recipient tissue. Individual transplanted cells were associated with each other and migrated chaotically with glial differentiation. Seven days after transplantation GFP+ cells were observed at different distances (600mkm, 1000mkm, 3000mkm) populating damaged zones at a rate of 90%, 56%, and 27% respectively. NSPC migration and differentiation in cell layers of neuroretina were higher (p<0.05) than for NSPC transplanted to the explant surface. In comparison, PE cells were available for migration and proliferation activity only after photoreceptor surface transplantation. The same data were obtained after NSPC and PE cell in vivo injection in Campbell rat eyes (intravitreal, retrobulbar and suprachoroidal transplantation of 50–100 thousands cells in 2µl).

Conclusions

Therefore the obtained data indicate: an effective in vitro model of explantation culture for detecting cell migration, differentiation and communication processes in recipient tissue directly after transplantation was developed and successfully applied for NSPC and MMSC in vitro transplantation. Using this technique it is possible to observe the reparation time for damaged tissue after SC transplantation; minimal cell-migration time to the damaged area; optimal transplantation time after damage; minimal number of transplanted SC required to start the reparation process; necessity of tissue micro surrounding for SC migration and choosing the best strategy for cell injection. Using this method it is possible to optimize clinical transplantation protocols and make them more effective.

Acknowledgements

This work is a realization of the Federal program for the years 2009–2013 entitled “Scientific and science-pedagogic staff of innovation Russia”.

References

1. Bull ND, Martin KR. Using stem cells to mend the retina in ocular disease. Regen Med. 2009 Nov;4(6):855-64. doi: 10.2217/rme.09.59.

2. Johansson K, Ehinger B. Structural changes in the developing retina maintained in vitro. Vision Res. 2005 Nov;45(25-26):3235-43. doi: 10.1016/j.visres.2005.05.022.

3. Kretz A, Hermening SH, Isenmann S. A novel primary culture technique for adult retina allows for evaluation of CNS axon regeneration in rodents. J Neurosci Methods. 2004 Jul 30;136(2):207-19. doi: 10.1016/j.jneumeth.2004.01.012.

4. Schrepfer S, Deuse T, Lange C, Katzenberg R, Reichenspurner H, Robbins RC, Pelletier MP. Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells. Stem Cells Dev. 2007 Feb;16(1):105-7. doi: 10.1089/scd.2006.0041.

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Introduction

Materials and methods

Results

Conclusions

Acknowledgements

This work is a realization of the Federal program for the years 2009–2013 entitled “Scientific and science-pedagogic staff of innovation Russia”.

References

1. Bull ND, Martin KR. Using stem cells to mend the retina in ocular disease. Regen Med. 2009 Nov;4(6):855-64. doi: 10.2217/rme.09.59.

2. Johansson K, Ehinger B. Structural changes in the developing retina maintained in vitro. Vision Res. 2005 Nov;45(25-26):3235-43. doi: 10.1016/j.visres.2005.05.022.

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Сергей А. Сергеев, Юлия В. Храмова

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Показана быстрая миграция трансплантированных НСПК и ММСК в эксплантатах сетчатки, поврежденных лазерным излучением (достоверно отличающаяся (p<0,05) от скорости миграции в интактных эксплантатах) в течение 24 часов, формирование ими нейритов и взаимосвязей с клетками сетчатки на протяжении 50 дней сокультивирования. Единичные клетки образовывали ассоциаты и экспрессировали GFAP. Через 7 дней после трансплантации на различных дистанциях (600, 1000, 3000мкм) от зоны повреждения детектировали 90%, 56%, 27% EGFP+ клеток соответственно. Для НСПК миграция была активней при инъекции вглубь слоев сетчатки, для ММСК достоверных отличий в миграции от способа инъекции выявлено не было.

Ключевые слова

стволовые клетки, трансплантация in vitro, органотипические культуры сетчатки

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Sergey A. Sergeev, Yulia V. Khramova

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Lomonosov MSU, Moscow, Russia

Correspondence
Lomonosov MSU, Moscow, Russia, Leninskie gory 1/12, 119234, Moscow, Russia; Phone: +79035187482
E-mail: embryossa@spam is badgmail.com

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Transplanted NSPC and MMSC quickly migrated in laser damaged retina explants (significantly differs (p<0.05) from the undamaged explants) during the first 24 hours, formed neuritis and connections with the retina cells, and were detected up to 50 days later in damaged recipient tissue. Individual transplanted cells were associated with each other and migrated chaotically with glial differentiation. Seven days after transplantation EGFP+ cells were observed in different distances (600 mkm, 1000 mkm, 3000 mkm) populating damaged zones in 90%, 56%, and 27% respectively. NSPC migration and differentiation in cell layers of neuroretina were higher (p<0.05) than for NSPC transplanted to the explant's surface, and no significant data was obtained for MMSC.

Keywords

Stem cells, in vitro transplantation, retina explant culture

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Sergeev, Yulia V. Khramova" ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(43) "

Sergey A. Sergeev, Yulia V. Khramova

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Keywords

Stem cells, in vitro transplantation, retina explant culture

Keywords

Stem cells, in vitro transplantation, retina explant culture

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Lomonosov MSU, Moscow, Russia

Correspondence
Lomonosov MSU, Moscow, Russia, Leninskie gory 1/12, 119234, Moscow, Russia; Phone: +79035187482
E-mail: embryossa@spam is badgmail.com

Lomonosov MSU, Moscow, Russia

Correspondence
Lomonosov MSU, Moscow, Russia, Leninskie gory 1/12, 119234, Moscow, Russia; Phone: +79035187482
E-mail: embryossa@spam is badgmail.com

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Сергей А. Сергеев, Юлия В. Храмова

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Единичные клетки образовывали ассоциаты и экспрессировали GFAP. Через 7 дней после трансплантации на различных дистанциях (600, 1000, 3000мкм) от зоны повреждения детектировали 90%, 56%, 27% EGFP+ клеток соответственно. Для НСПК миграция была активней при инъекции вглубь слоев сетчатки, для ММСК достоверных отличий в миграции от способа инъекции выявлено не было. <h3>Ключевые слова</h3> стволовые клетки, трансплантация in vitro, органотипические культуры сетчатки " ["TYPE"]=> string(4) "HTML" } ["DESCRIPTION"]=> string(0) "" ["VALUE_ENUM"]=> NULL ["VALUE_XML_ID"]=> NULL ["VALUE_SORT"]=> NULL ["~VALUE"]=> array(2) { ["TEXT"]=> string(1445) "

Ключевые слова

стволовые клетки, трансплантация in vitro, органотипические культуры сетчатки

Ключевые слова

стволовые клетки, трансплантация in vitro, органотипические культуры сетчатки

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Introduction

Hematopoietic stem cell (HSC) transplantation is the best example of cell technology usage in clinical practice. However, the complexity associated with low HSC engraftment, the development of the "graft-versus-host disease" reaction, and infectious complications have not been eliminated so far [24]. The causes and manifestations of immunological disorders are clear in general, but low engraftment etiology remains the subject of lively debates. The subject of the development is progenitor cells, conditioning protocols, and the "destiny" of elements of bone marrow (BM) stroma transplanted along with HSCs, which specifications are necessary for the development of HSC engraftment. Our research group efforts are also focused on solving the problem. Thus this essay is directed toward systemizing the fundamental and current data of HSC engraftment and the causes of its low level in allogeneic transplantation, as well as to discuss world trends in optimization of HSC engraftment using one’s own data.

Current approaches to improve HSC engraftment

Modern solutions aimed at increasing HSC engraftment can be divided into two directions:
a) techniques directed to the donor’s body and the graft in order to improve the quality and graft "acceptance";
b) methods of influence on the recipient’s body to optimize the "acceptance" of the transplanted HSCs.

The first direction is based on current data of possible HSC expansion in vitro [23], cell activation, and on studies of alternative HSC sources (cord blood, in particular) [5]. The fundamentals of the second approach are associated with studies of BM stroma, HSCs niches, and the processes occurring to stroma cellular elements forming a part of the graft into the recipient’s body. There are no comprehensive data of the processes occurring to BM stroma or bone tissue as an element of cell niches under the influence of conditioning protocols, and there is no generalized concept of the "destiny" of transplanted cellular elements of BM stroma. At the same time the necessity for such data is enormous, as it, on the one hand, helps to develop the most effective methods of HSC engraftment, and on the other hand helps to develop the technology of pathology correction of connective tissues (myelofibrosis, osteogenesis imperfecta, etc).

The concept of HSC niches is the best implementation of theoretical data on BM stroma and its role in providing hematopoiesis, and it serves for better understanding and practical application as well.

HSC niches: fundamental provisions and new vision

Generally, a cell niche is an elementary functional unit of the microenvironment. However, there is no unified, generally accepted definition of the "cell niche". The first part of the hypothetical definition, reflecting niche structure (the set of spatially organized cell elements and components of intercellular matrix) is not contested, although the functional aspect is debatable. Some authors believe that a cell niche maintains a stem cell in undifferentiated quiescent state [3]. The majority take a different view, giving a cell niche a greater role: not only the maintenance of "cellular homeostasis", but also the regulation and provision of morpho-functional activity [16].

Nowadays, scientists distinguish two types of HSCs niches: vascular (endothelial) and endosteal (osteoblastic) [22]. To my mind, this differentiation is rather conditional, since the osteoblastic line cells, forming the niches of the same name and requiring active oxygenation, are situated close to the microvasculature (the critical distance is 200–250 mkm [11]). Consequently, sinusoidal endothelial cells (EC) must be an integral component of the so-called osteoblastic niches. The main cellular elements of BM niches are osteoblasts, EC and multipotent mesenchymal stromal cells (MMSCs) [16]. Implementation of the activity of cells which form cell niches is carried out in two principal directions.

A. Direct impact due to intercellular contacts and products of specific biologically active substances (SDF-1, SCF, TPO, interleukins, etc) [17]. Thus, spindle-shaped osteoblasts by means of tight adhesive contacts with N-cadherin/β-catenin system interact with HSCs, maintaining them in a "quiescent" state [3, 10];

B. Indirect impact: paracrine incentives transfer (erythropoietin, SDF-1, parathyroid hormone, etc.) from other components of the uniform functional system of hematopoiesis: kidneys, liver, spleen, endocrine glands, autonomic nervous system [25, 26].

However, the results of studies in recent years significantly extend the notion of HSC niches, particularly, HSCs that are found in adipose tissue [13], spleen [15], and placenta [12], given the description of HSC migration to extramedullary tissues, for instance, in polytrauma [1]. Taken together, the results of HSCs' persistence in extramedullary tissues indicate the presence of their niches outside the BM. In this respect, the fundamental classic works of the Russian histology school become critical again. According to the theory of "mesenchymal reserve" by A. Maximow, there are progenitor cells for the connective tissues in definitive tissues, marked by him as "stem mesenchymal cells" [19]. Later, the theory was supported by A. Zavarzin [2] and N. Khlopin [4] and has now been proven with contemporary data [6]. The presence of poorly differentiated cells (perivasculоcytes) going along with blood vessels conforming by their differentiating MMSC potency is now proven and indisputable.

Thus, there is at least a vascular HSC niche in extramedullary tissues. In light of fundamental notions and current data, the adjustment of concepts of HSC niches seems to be logical. To my mind, it is correct to speak about a uniform cell niche consisting of two components (Table 1).

Table 1. Components of HSC niches

Signs of comparison	Nonspecific component	Specific component
Cells	EC and MMSCs	Resident cells of the definitive tissues (osteoblasts of bone marrow, fibroblasts of fibrous connective tissue, etc.)
Function	Provision of migration, transduction of regulatory incentives and regulation of non-specific functions	Provision of a specific function*
*Osteoblasts play the defining role in the regulation of hematopoietic HSC function. It is proved that osteoblasts induce proliferation and differentiation of HSCs in a greater degree than MMSCs [20]. In extramedullary tissues the specific component of cell niches incentivizes HSC homing and the implementation of other specific effects, such as participation in reparative regeneration [1].
Cell Ther Transplant. 2011;3:e.000095.01. doi:10.3205/ctt-2011-en-000095-table1

Such a systematic generalized concept of HSCs niche is easier and more practical.

The possibilities to restore cell niches

The practical significance of the concept of HSCs niche is in clearer comprehension of the functioning of BM stroma and the possibility of a selective approach to its restoration after conditioning. However, it is necessary to solve two problem issues to perform it in practice.
1. What components of cell niches are damaged with different conditioning protocols, and to what degree?
2. What way can a damaged component of a cell niche be restored?

Currently there are not enough data to give an exhaustive answer to the questions. It is established that in total irradiation the specific component of BM niches is not really damaged, thus providing a moderate level of HSC engraftment [10]. Irradiation causes marked damage to a nonspecific component: sinusoidal ES are damaged [14], and MMSCs obtain functional defects [21]. Apparently, a similar situation is typical of extramedullary HSCs niches. The influence of chemotherapy on BM niches is less studied due to the great variety of protocols. In general chemotherapy does less damage to the nonspecific component of niches [14].

However, taking into consideration the paucity of data on the damaging effect of conditioning regimens, therapeutic conditions which are aimed at restoring the two components of the niches are clear.

Our research group managed to figure out the processes to a certain extent, concerning the transplanted cellular elements of BM stroma. It has been shown with an experimental model of osteogenesis that the restoration of wholeness of mice shin bones irradiated at a dose of 7–7.5 Gy is mainly carried out due to the transplanted transgenic (GFP+) cells of allogeneic BM (Fig. 1). Moreover, even under physiological conditions the inserted components of allogeneic BM stroma planted the patches of typical location of the dispersed cambium of skeletal tissues (periosteum, endosteum, BM stroma, perivascular niches). Up to 30% of osteocytes of bone tissue were of donor origin (Fig. 2) [7]. These data are consistent with the results of other research groups and prove the possibility of the planting of recipient tissues transplanted with HSC elements of allogeneic BM stroma, as well as confirm their involvement in the implementation of reparative processes in skeletal tissues [9]. Consequently, the use of components of BM stroma (MMSCs, endothelial progenitor cells) is potentially practiced for regeneration of BM HSC niches, as well as for treatment of patients with the pathology of skeletal tissue stroma. However, the number of necessary stroma components in the whole BM is not enough (2–5×10³ MMSCs/kg), and consequently only 7% of transplanted MMSCs remain for long in the stroma of a recipient’s BM [21]. At the same time the expansion of MMSCs in vitro up to the concentration in the graft (20–30×10⁶ MMSCs per 1 kg of a recipient’s weight) significantly increases the level of stroma planting of the recipient’s BM, which are maintained over time [8]. Moreover, there are published data on the improvement of HSCs' engraftment during their co-transplantation with so-called "stromal cells of umbilical cord blood" [18].

Figure 1. Shin bone tissues of the recipient mouse: A: spongy bone tissue (1: GFP-positive cells of bone marrow and stroma, 2: endosteum with GFP-positive osteoblast, 3: bony trabeculae), magnification ×100. B: trabecula of bone and cartilage regenerate, consisting of GFP-positive cells; magnification ×400. C: isogenous group of GFP-positive chondrocytes in the bone and cartilage regenerate; magnification ×800. Confocal microscopy. Color: propidium iodide

Figure 2. Osteocyte in the reticular fibrous bone tissue of the regenerate, having donor origin: А: a cell nucleus, propidium iodide stained, B: cytoplasm autofluorescence; C: combined image. Magnification ×800. Confocal microscopy

Thus, the fortification of the whole BM graft or a purified HSCs fraction with stromal cellular components in concentrations exceeding physiologic congestion is potentially effective. Furthermore, considering the important role of EC as the components of HSCs niches, transplantation of their progenitor cells in high concentrations seems to be promising, especially in combination with factors stimulating angiogenesis (for instance, medicines on the basis of VEGF). In any case, the actual clinical application of the methods requires further clinical trials.

Acknowledgements

The author wishes to thank Roman V. Deev (OJSC "Human Stem Cells Institute", Moscow, Russia), and Natalia V. Tsupkina (Institute of Cytology RAS, St. Petersburg, Russia), for qualified direction and assistance in scientific research.

References

1. Александров ВН, Сергеев ВС. Влияние тяжелой политравмы на миграцию стволовых кроветворных клеток у мышей. Клеточная трансплантология и тканевая инженерия. 2006;2(4):59-62.

2. Заварзин АА. Курс гистологической и микроскопической анатомии. Л.: Гос. из-во мед. лит-ры. 1938. 634 c.

3. Пинаев ГП. Ниши стволовых клеток (понятие «тканевой ниши» и значение белков внеклеточного матрикса для регулирования функций стволовых клеток). Материалы школы-конференции для молодых ученых «Клеточные технологии для регенеративной медицины». СПб. 17-21 окт. 2011.

4. Хлопин НГ. Общебиологические и экспериментальные основы гистологию Л.: Из-во АН СССР. 492 с.

5. Chou S, Chu P, Hwang W, Lodish H. Expansion of human cord blood hematopoietic stem cells for transplantation. Cell Stem Cell. 2010 Oct 8;7(4):427-8. doi: 10.1016/j.stem.2010.09.001.

6. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008 Sep 11;3(3):301-13. doi: 10.1016/j.stem.2008.07.003.

7. Deev RV, Tsupkina NV, Serikov VB, Gololobov VG, Pinaev GP. Participation of transfused bone marrow cells in reparative osteohistogenesis. Tsitologiia. 2005;47(9):755-759. In Russian.

8. Devine SM, Bartholomew AM, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol. 2001 Feb;29(2):244-55.

9. Dominici M, Pritchard C, Garlits JE, Hofmann TJ, Persons DA, Horwitz EM. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc Natl Acad Sci U S A. 2004 Aug 10;101(32):11761-6. doi: 10.1073/pnas.0404626101.

10. Dominici M, Rasini V, et al. Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation. Blood. 2009 Sep 10;114(11):2333-43. doi: 10.1182/blood-2008-10-183459.

11. Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med. 1973 Oct 1;138(4):745-53.

12. Gekas C, Dieterlen-Lièvre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell. 2005 Mar;8(3):365-75. doi: 10.1016/j.devcel.2004.12.016.

13. Han J, Koh YJ, Moon HR, Ryoo HG, Cho CH, Kim I, Koh GY. Adipose tissue is an extramedullary reservoir for functional hematopoietic stem and progenitor cells. Blood. 2010 Feb 4;115(5):957-64. doi: 10.1182/blood-2009-05-219923.

14. Hooper AT, Butler JM, et al. Engraftment and Reconstitution of Hematopoiesis Is Dependent on VEGFR2-Mediated Regeneration of Sinusoidal Endothelial Cells. Cell Stem Cell. 2009 Mar 6;4(3):263-74. doi: 10.1016/j.stem.2009.01.006.

15. Iseki A, Morita Y, Nakauchi H, et al. Hematopoietic Stem Cells in the Mouse Spleen. Blood (ASH Annual Meeting Abstracts). 2008:112.

16. Isern J, Méndez-Ferrer S. Stem cell interactions in a bone marrow niche. Curr Osteoporos Rep. 2011 Dec;9(4):210-8.

17. Li T, Wu Y. Paracrine molecules of mesenchymal stem cells for hematopoietic stem cell niche. Bone Marrow Res. 2011;2011:353878. doi: 10.1155/2011/353878.

18. Liu Y, Yi L, Zhang X, Gao L, Zhang C, Feng YM, Chen XH. Cotransplantation of human umbilical cord blood-derived stromal cells enhances hematopoietic reconstitution and engraftment in irradiated BABL/c mice. Cancer Biol Ther. 2011 Jan 1;11(1):84-94.

19. Brodersen J, Maximow A, Schaffer J. Handbuch der mikroskopischen Anatomie des Menschen. Bd. 2., Die Gewebe; T. 1., Epithel- u. Drüsengewebe, Bindegewebe und blutbildende Gewebe, Blut. Berlin: Springer, 1927.

20. Mishima S, Nagai A, Abdullah S, Matsuda C, Taketani T, Kumakura S, Shibata H, Ishikura H, Kim SU, Masuda J. Effective ex vivo expansion of hematopoietic stem cells using osteoblast-differentiated mesenchymal stem cells is CXCL12 dependent. Eur J Haematol. 2010 Jun;84(6):538-46. doi: 10.1111/j.1600-0609.2010.01419.x.

21. Morikawa S, Mabuchi Y, et al. Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow. J Exp Med. 2009 Oct 26;206(11):2483-96. doi: 10.1084/jem.20091046.

22. Morrison SJ, Spradling AC. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell. 2008 Feb 22;132(4):598-611. doi: 10.1016/j.cell.2008.01.038.

23. Nishino T, Wang C, Mochizuki-Kashio M, Osawa M, Nakauchi H, Iwama A. Ex vivo expansion of human hematopoietic stem cells by garcinol, a potent inhibitor of histone acetyltransferase. PLoS One. 2011;6(9):e24298.

24. Rafeah NT, Fadilah SA. The A-B-C of haematopoietic stem cell transplantation. Med J Malaysia. 2009 Mar;64(1):94-100.

25. Rashidi N, Adams GB. The influence of parathyroid hormone on the adult hematopoietic stem cell niche. Curr Osteoporos Rep. 2009 Jul;7(2):53-7.

26. Visigalli I, Biffi A. Maintenance of a functional hematopoietic stem cell niche through galactocerebrosidase and other enzymes. Curr Opin Hematol. 2011 Jul;18(4):214-9.

" ["~DETAIL_TEXT"]=> string(22701) "

Introduction

Current approaches to improve HSC engraftment

HSC niches: fundamental provisions and new vision

Signs of comparison	Nonspecific component	Specific component
Cells	EC and MMSCs	Resident cells of the definitive tissues (osteoblasts of bone marrow, fibroblasts of fibrous connective tissue, etc.)
Function	Provision of migration, transduction of regulatory incentives and regulation of non-specific functions	Provision of a specific function*
*Osteoblasts play the defining role in the regulation of hematopoietic HSC function. It is proved that osteoblasts induce proliferation and differentiation of HSCs in a greater degree than MMSCs [20]. In extramedullary tissues the specific component of cell niches incentivizes HSC homing and the implementation of other specific effects, such as participation in reparative regeneration [1].
Cell Ther Transplant. 2011;3:e.000095.01. doi:10.3205/ctt-2011-en-000095-table1

Such a systematic generalized concept of HSCs niche is easier and more practical.

The possibilities to restore cell niches

Acknowledgements

References

2. Заварзин АА. Курс гистологической и микроскопической анатомии. Л.: Гос. из-во мед. лит-ры. 1938. 634 c.

4. Хлопин НГ. Общебиологические и экспериментальные основы гистологию Л.: Из-во АН СССР. 492 с.

5. Chou S, Chu P, Hwang W, Lodish H. Expansion of human cord blood hematopoietic stem cells for transplantation. Cell Stem Cell. 2010 Oct 8;7(4):427-8. doi: 10.1016/j.stem.2010.09.001.

6. Crisan M, Yap S, Casteilla L, et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell. 2008 Sep 11;3(3):301-13. doi: 10.1016/j.stem.2008.07.003.

7. Deev RV, Tsupkina NV, Serikov VB, Gololobov VG, Pinaev GP. Participation of transfused bone marrow cells in reparative osteohistogenesis. Tsitologiia. 2005;47(9):755-759. In Russian.

8. Devine SM, Bartholomew AM, et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol. 2001 Feb;29(2):244-55.

11. Folkman J, Hochberg M. Self-regulation of growth in three dimensions. J Exp Med. 1973 Oct 1;138(4):745-53.

12. Gekas C, Dieterlen-Lièvre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell. 2005 Mar;8(3):365-75. doi: 10.1016/j.devcel.2004.12.016.

15. Iseki A, Morita Y, Nakauchi H, et al. Hematopoietic Stem Cells in the Mouse Spleen. Blood (ASH Annual Meeting Abstracts). 2008:112.

16. Isern J, Méndez-Ferrer S. Stem cell interactions in a bone marrow niche. Curr Osteoporos Rep. 2011 Dec;9(4):210-8.

17. Li T, Wu Y. Paracrine molecules of mesenchymal stem cells for hematopoietic stem cell niche. Bone Marrow Res. 2011;2011:353878. doi: 10.1155/2011/353878.

22. Morrison SJ, Spradling AC. Stem cells and niches: mechanisms that promote stem cell maintenance throughout life. Cell. 2008 Feb 22;132(4):598-611. doi: 10.1016/j.cell.2008.01.038.

24. Rafeah NT, Fadilah SA. The A-B-C of haematopoietic stem cell transplantation. Med J Malaysia. 2009 Mar;64(1):94-100.

25. Rashidi N, Adams GB. The influence of parathyroid hormone on the adult hematopoietic stem cell niche. Curr Osteoporos Rep. 2009 Jul;7(2):53-7.

26. Visigalli I, Biffi A. Maintenance of a functional hematopoietic stem cell niche through galactocerebrosidase and other enzymes. Curr Opin Hematol. 2011 Jul;18(4):214-9.

Correspondence
Ilya Ya. Bozo, Moscow Dental University, OJSC "Human Stem Cells Institute", 3/2 Gubkina str., 119991, Moscow, Russia; Phone: +7965147-12-77, Fax: +7495646-80-76
E-mail: bozo.ilya@gmail.com

The essay presents the problematic aspects of hematopoietic stem cell transplantation, from the position of fundamental notions about cell niches and contemporary data on the bone marrow stroma of recipient and donor, taking into account the results of one’s own research.

Keywords

hematopoietic stem cells (HSCs) transplantation, cell niches, bone marrow stroma

В эссе представлены проблемные аспекты трансплантации гемопоэтических стволовых клеток, возможности увеличения энграфмента с позиции фундаментальных представлений о клеточных нишах и современных данных о строме костного мозга реципиента и донора с учетом результатов собственных исследований.

Ключевые слова

трансплантация гемопоэтических стволовых клеток, клеточные ниши, строма костного мозга

Keywords

hematopoietic stem cells (HSCs) transplantation, cell niches, bone marrow stroma

Keywords

hematopoietic stem cells (HSCs) transplantation, cell niches, bone marrow stroma

Ключевые слова

трансплантация гемопоэтических стволовых клеток, клеточные ниши, строма костного мозга

Ключевые слова

трансплантация гемопоэтических стволовых клеток, клеточные ниши, строма костного мозга

Introduction

Muscular dystrophies are a highly heterogeneous group of inherited disorders characterized by progressive skeletal muscle weakness and necrosis of the muscle tissue. Currently, more than 20 different monogenous disorders, inherited in autosomal recessive, autosomal dominant, and X-linked ways, are referred to as muscular dystrophies. Their incidence varies from 1 in 3000 boys for Duchenne Muscular Dystrophy (DMD) to 1 in 200 000 for LGMD2B [9, Jain Foundation Data]. The onset of the inherited muscular dystrophies, as well as their natural history also can be very different. They can start in early childhood and rapidly develop, causing death in the early twenties as happens in DMD [1], or have an average onset in the late twenties, and mildly progress, not affecting the average lifespan, as for most LGMDs. This group of diseases is easily recognized, however differential diagnosis poses some issues for a physician, as in most cases it should be based on molecular and DNA sequence data. There is no specific treatment for any of the forms of muscular dystrophy. Physiotherapy and aerobic exercises may help to prevent contractures and maintain muscle tone, orthoses may be needed to improve quality of life in some cases, but no actual long-term therapy has been found [1]. Currently the two most promising approaches to treat muscular dystrophies in general, and DMD in particular, are gene and cell therapy or their combination. This short review highlights the key points in these fields, as well as main expectations and pitfalls in gene therapy of Duchenne myodystrophy.

Gene therapy for DMD

Muscular dystrophies, like all inherited monogenous diseases, are caused by mutations in various genes, and, thus, gene therapy is a very promising approach to their treatment. Most of the gene therapy studies in this field have been focused on Duchenne Muscular Dystrophy, one of the most widespread and fatal neuro-muscular diseases. Therefore, this particular condition is a very good model, which should help to elucidate the main issues, hopes, and pitfalls in the field.

DMD is caused by a vast spectrum of mutations, mainly the large-span deletions of the exons in the 2.3Mb dystrophin (DMD) gene [6, 13]. The dystrophin mRNA spans nearly 1000kb and is primarily translated in the muscle tissue, where the protein of the same name plays a major role in muscle fiber contraction and maintenance. Mutations in the dystrophin gene impair normal protein synthesis, causing the consequent progressive myofiber necrosis resulting in progressive muscle weakness. Symptoms usually appear in male children before age 5 and may be visible in early infancy. Progressive proximal muscle weakness of the legs and pelvis associated with a loss of muscle mass is observed first. Eventually this weakness spreads to the arms, neck, and other areas.

Given this, the ideal vector for DMD gene therapy should be able to carry a 1000 kb of DNA and easily penetrate muscle fibers, and then effectively express functional dystrophin for an unlimited period of time. Moreover, it ought to be safe (non-mutagenic or immunogenic) and its expression should be easily regulated. But for the carried DNA size, these requirements could be applied to the ideal vector to treat any muscular dystrophy. In this connection, let’s try to evaluate the most promising vector types, taking into account the proposed criteria of safety and efficiency.

Currently all gene therapy delivery systems can be divided into two large groups: viral and non-viral. Viral vectors are the most commonly used: during the period from 1989 to 2010 they were employed in more than 60% of gene therapy clinical trials (Wiley Interscience Gene Therapy Trials Worldwide Database; Edelstein et al., 2007). Their major advantages are high effectiveness and specificity of transgene delivery.

Amongst the viral vectors, only lentiviruses and adeno-associated viruses (AAV) can be effectively used for muscular dystrophy's gene therapy, as only they are able to efficiently penetrate the myofibers and express the transgene there. The use of lentiviruses, despite their high transgene expression efficiency and relative safety, is connected with side effects that are too serious for them to be put into clinical practice. For instance, patients who underwent such gene therapy demonstrated a HIV-positive profile [3].

The AAVs are the safest viral vectors due to their low immunogenicity, the absence of the insertional mutagenesis risk and other side effects. In addition, they are one of the most effective: stable transgene expression in primates could be detected even 18 months after injection [10]. The major issue in the use of this vector type for DMD gene therapy is the very small size of the therapeutic cassette — only 4 kb [15]. However, this has been solved by the use of a truncated version of the gene (“mini-dystrophin”) instead of the complete one. This approach efficiently helped to ameliorate the disease, but not to cure, however it successfully passed through the Phase I clinical trial [5]. Unfortunately, is inapplicable for most of the muscular dystrophies, as not all the causative genes have “mini” versions.

Thus, none of the viral vectors, despite their efficiency and safety, fit the ideal criteria, for different reasons: the small size of the therapeutic cassette in the case of AAV, serious side effects in lentiviruses, and inefficiency of the other extensively studied viral vector types.

Non-viral vectors, such as plasmids and human artificial chromosomes (HACs), do have advantages over the viral ones, because of their safety and theoretically unlimited size of the therapeutic cassette. However, the efficiency of the plasmid-mediated transgene delivery is rather poor as well as the time of its expression [for instance, see 16], especially when it is delivered systemically.

HACs on the other hand are able to maintain expression of genomic-sized transgenes within target cells for an unlimited time. What's more, they do not need to integrate into the host genome to maintain the transgene expression, and the latter one’s level is regulated by the intracellular mechanisms. In spite of the fact that their construction and delivery pose certain technical challenges, the recent advances in this field are very promising [4]. Moreover, the first successful results of the preclinical trial of the cell-based therapy exploiting HACs bearing DMD in mice have recently been published by Tedesco et al., 2011. The development of this strategy to treat other muscular dystrophies is a way to find the cure for this group of diseases.

So, non-viral vectors, and HACs in particular, represent the most promising direction in the DMD treatment research.

Cell therapy for muscular dystrophies

The main issue in developing an effective therapy for muscular dystrophies is that the muscle tissue is a non-dividing one, and its reparation is a very complicated and relatively rarely studied process. Even if one manages to find an effective and safe way to transfer genes to the muscle fiber and to obtain a stable transgene expression in the target cell, the results of such gene therapy aren't very promising. For instance, the presence of the transgenic dystrophin in the myofibrils will ameliorate the DMD and will prevent the disease progress, but will not help to restore them. Therefore such therapy only makes sense in the early stages of the disease.

The most effective way to solve this issue is to combine gene and cell therapy by introducing genetically modified stem cells, so that the restored myofibrils effectively express the transgene to prevent their damage and necrosis. However, not many types of stem cells fit the criteria necessary for the stem cell therapy of muscular dystrophies. Such cells should effectively fuse with the myofibers, they should be easily obtained from the patient’s tissue and then be easily expanded in vitro, and finally they must be systemically deliverable. For instance, mesoangioblasts, currently one of the most promising cell types to treat DMD are easily cultivated, effectively fuse with the myofibers and can be delivered by intravenous infusion, but can only be harvested in the very early stages of perinatal development [11]. They could be obtained from an allogenic donor, but this poses the question of their availability, as well as ethical issues. Other populations of myogenic stem cells, such as CD133+ satellite cells, are very hard to obtain, and the use of embryoniс stem cells and iPS cells raises ethical and safety issues respectively [7].

However there are two groups of the myogenic stem cells which partially suit all the criteria mentioned — these are CD133+ hematopoietic (HSC) and mesenchymal stem cells (MSC), which can be easily obtained from a patient, expanded in vitro, and have been shown to contribute to muscle regeneration [2, 14]. Currently, the therapeutic potential of these two stem cells populations are being extensively studied.

Modification of these cell types with HACs bearing the necessary gene and their consequent transplantation to the patient via IV injection could be a very promising strategy to treat first DMD, and then muscular dystrophies in general. Thus, the use of HAC-modified autologous CD133+ HSC or MHC opens new perspectives for the treatment of the muscular dystrophies. However, much research must be done to bring this technology from paper into clinical practice.

Acknowledgements

The author is a project manager of OJCL “Human Stem Cells Institute”, Moscow, Russian Federation.

References

1. Boland BJ, Silbert PL, Groover RV, Wollan PC, Silverstein MD. Skeletal, cardiac, and smooth muscle failure in Duchenne muscular dystrophy. Pediatr Neurol. 1996 Jan;14(1):7-12.

4. Kazuki Y, Oshimura M. Human artificial chromosomes for gene delivery and the development of animal models. Mol Ther. 2011 Sep;19(9):1591-601. doi:10.1038/mt.2011.136.

8. Nelson, MR Pediatrics. Rehabilitation medicine quick reference. New York: Demos Medical, 2010. 268 pp. ISBN 978-1-933864-60-0.

11. Sampaolesi M, Torrente Y, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science. 2003;301(5632):487-92.

12. Tedesco FS, Hoshiya H, et al. Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy. Sci. Transl. Med. 2011;3(96):96ra78. doi: 10.1126/scitranslmed.3002342.

13. Tennyson CN, Klamut HJ, Worton RG. The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nature Genet. 1995;9:184-190. doi: 10.1038/ng0295-184.

Introduction

Gene therapy for DMD

Cell therapy for muscular dystrophies

Acknowledgements

The author is a project manager of OJCL “Human Stem Cells Institute”, Moscow, Russian Federation.

References

1. Boland BJ, Silbert PL, Groover RV, Wollan PC, Silverstein MD. Skeletal, cardiac, and smooth muscle failure in Duchenne muscular dystrophy. Pediatr Neurol. 1996 Jan;14(1):7-12.

4. Kazuki Y, Oshimura M. Human artificial chromosomes for gene delivery and the development of animal models. Mol Ther. 2011 Sep;19(9):1591-601. doi:10.1038/mt.2011.136.

8. Nelson, MR Pediatrics. Rehabilitation medicine quick reference. New York: Demos Medical, 2010. 268 pp. ISBN 978-1-933864-60-0.

11. Sampaolesi M, Torrente Y, et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science. 2003;301(5632):487-92.

12. Tedesco FS, Hoshiya H, et al. Stem cell-mediated transfer of a human artificial chromosome ameliorates muscular dystrophy. Sci. Transl. Med. 2011;3(96):96ra78. doi: 10.1126/scitranslmed.3002342.

13. Tennyson CN, Klamut HJ, Worton RG. The human dystrophin gene requires 16 hours to be transcribed and is cotranscriptionally spliced. Nature Genet. 1995;9:184-190. doi: 10.1038/ng0295-184.

Мышечные дистрофии представляют собой весьма гетерогенную группу наследственных заболеваний, характеризующихся прогрессирующей мышечной слабостью и некрозом мышечной ткани. В настоящее время не имеется специфического лечения для любых форм мышечной дистрофии. В этой области большинство работ по генной и клеточной терапии сконцентрировано на мышечной дистрофии Дюшенна (ДМД) – одном из наиболее частых нервно-мышечных заболеваний со смертельным исходом. В данном кратком обзоре обозначены ключевые темы в этой области, а также основные надежды и препятствия, касающиеся генной терапии ДМД.

Ключевые слова

миодистрофия Дюшенна, генная терапия, искусственные человеческие хромосомы

Muscular dystrophies are a highly heterogeneous group of inherited disorders characterized by progressive skeletal muscle weakness and necrosis of the muscle tissue. Currently there is no specific treatment for any of the forms of muscular dystrophy. Most of the gene and cell therapy studies in this field have focused on Duchenne Muscular Dystrophy (DMD), one of the most frequent and fatal neuro-muscular diseases. This short review highlights the key points in these field, as well as main expectations and pitfalls concerning the gene therapy of DMD.

Keywords

Muscular dystrophies are a highly heterogeneous group of inherited disorders characterized by progressive skeletal muscle weakness and necrosis of the muscle tissue. Currently there is no specific treatment for any of the forms of muscular dystrophy. Most of the gene and cell therapy studies in this field have focused on Duchenne Muscular Dystrophy (DMD), one of the most frequent and fatal neuro-muscular diseases. This short review highlights the key points in these field, as well as main expectations and pitfalls concerning the gene therapy of DMD.

Keywords

Muscular dystrophies are a highly heterogeneous group of inherited disorders characterized by progressive skeletal muscle weakness and necrosis of the muscle tissue. Currently there is no specific treatment for any of the forms of muscular dystrophy. Most of the gene and cell therapy studies in this field have focused on Duchenne Muscular Dystrophy (DMD), one of the most frequent and fatal neuro-muscular diseases. This short review highlights the key points in these field, as well as main expectations and pitfalls concerning the gene therapy of DMD.

Keywords

Ключевые слова

миодистрофия Дюшенна, генная терапия, искусственные человеческие хромосомы

Ключевые слова

миодистрофия Дюшенна, генная терапия, искусственные человеческие хромосомы

Introduction

Autologous bone marrow mononuclear stem cells (BMMC) have been used in clinical practice for more than nine years [13]. It has been proven proved that this therapy is effective in patients with dilated cardiomyopathy [5, 12] and patients with severe heart failure [1, 3, 14]. However, there have been very few studies on the usage of autologous bone marrow mononuclear stem cells in patients with severe angina pectoris when it is impossible to use mechanical revascularization (percutaneous coronary intervention: PCI, or coronary artery bypass grafting: CABG), or it is refractory to conventional medical therapy [8, 11, 16]. This group includes patients with distal coronary atherosclerotic damage, patients who received angioplasty and stenting with only part of involvement of the coronary artery, and patients with a relapse of angina pectoris after CABG. The last group of patients is the largest because it is known that only 38-45% of venous grafts are patent after ten years of operation [10]. The number of patients who have a relapse of angina pectoris after CABG increases in proportion to the number of operations.

Patients with severe heart failure or low left ventricle ejection fraction were excluded from our clinical trial.

Patients and Methods

The BMMC group consisted of 17 patients who underwent autologous bone marrow mononuclear stem intracoronary infusion between 2008 and 2010. A control group consisted of 10 patients. A comparative breakdown of both groups is presented below (Table 1):

Table 1. A comparative breakdown of the BMMC and the control groups

Indice	BMMC group (n=17)	Control group (n=10)
Male	14 (83%)	9 (90%)
Female	3 (17%)	1 (10%)
Median age	60.2±9	62.5±7
Previous myocardial infarction	1.2±0,8	1.4±0,7
Functional class of angina pectoris (CCS)	2.9±0,4	2.3± 0,5
Exercise test (Mets)	4.6	4.6
Nitroglycerin usage (pill/week)	28	7
Arterial hypertension	12 (70%)	5 (50%)
Diabetes mellitus	2 (12%)	1(10%)
Smoking	7 (41%)	3 (30%)
Cholesterol (mmol/l)	5.5	4.6
Relapse angina pectoris after CABG	6 (35%)	4 (40%)
Distal coronary atherosclerotic damage	8 (47%)	----
Incomplete myocardial revascularization (PCI)	3 (18%)	6 (60%)
LVIDd (mm)	51.6±2	52±4
LVIDs (mm)	35.8± 2	36.5±4
LVEF (%)	57.2±4	60.3±5
One coronary vessel disease	4 (23,5%)	6 (60%)
Two coronary vessel diseases	9 (53%)	3 (30%)
Three coronary vessel diseases	4 (23.5%)	1 (10%)
Cell Ther Transplant. 2011;3:e.000097.01. doi:10.3205/ctt-2011-en-000097-table1

It is seen that groups are comparable in sex, age, concomitant disease, and left ventricle injection fraction. There are more patients with two and thee coronary vessel diseases in the BMMC group, which explains the higher rate of angina pectoris. All of patients received optimal medical treatment, which was fixed before and was not changed during the research period. All patients were examined before and after the research period. Single-photon emission computed tomography was performed to estimate myocardial perfusion. Questionnaire SF-36 was used for an estimation of the patient’s quality of life.

Results

During the research period one patient has died in each group, for a mortality rate of 6% in the BMMC group and 10% in the control group. The functional rate of angina pectoris (CCS) has showed an improvement in the BMMC group compared with the control group. The median of Canadian Cardiovascular Society class decreased from class 3 to 2 in the BMMC group (p<0.001) and increased in the control group from class 2 to 3 (p>0.05). However, control group contained more patients who received percutaneous coronary intervention. We showed that the median of exercise time rose from 4.6 to 7.0 Mets in the treatment group (p>0.05), while there were no changes in the control group (p>0.05). Median nitroglycerine usage fell from 28 to 7 pills of nitroglycerine per week (p<0.05) in the treatment group and did not change in the control group over one year (p>0.05). There were not arrhythmias in either group.

The table 2 shows versions of answers from the SF-36 questionnaire that patients gave for the question: “Compared to one year ago, how would you rate your health in general now?”:

Table 2. Versions of answers from the SF-36 questionnaire

Answers	Main group (N=17)	Control group (N=10)
Much better now	7 (35 %)	1 (10%)
Somewhat better now	8 (53 %)	-
About the same	2 (12 %)	5 (50 %)
Somewhat worse now	-	4 (40 %)
Much worse now	-	-
Cell Ther Transplant. 2011;3:e.000097.01. doi:10.3205/ctt-2011-en-000097-table2

It can be seen that 88% of patients from the BMMC group felt better one year after the intracoronary infusion of autologous bone marrow mononuclear stem cells. In the control group only 1 person (10%) felt better, a half the patients did not notice any changes, and 40% felt worse than a year before.

Baseline scores of the eight SF-36 scales were approximately the same in both groups before the study. At the end of one year a great increase in quality of life in the BMMC group can be observed (Fig. 1). The best result it is seen in the Physical Functioning (PF) scale, which describes exercise times of the patients, and the Role-Physical (RP) scale, which describes the influence of physical condition on our daily work.

We found a strong correlation between the value of the Bodily Pain (BP) scale and the Canadian Cardiovascular Society class. (Spirman’s coefficient of rank data analysis = 0756, p=0.48). It is seen that the Bodily Pain scale went up in the BMMC group:

In the control group it is not seen considerably increasing in all scales and the scale of Bodily Pain has changed in opposite direction, which correlates well with an increase of Canadian Cardiovascular Society class in the control group.

A significant improvement in myocardial perfusion measured by single-photon emission computed tomography was noticed in the BMMC group; the median hypo perfusion zone decreased from 13.5 to 10.1 % (p>0.05).

Discussion

Intracoronary infusion of autologous bone marrow mononuclear stem cells decreases such parameters as the functional class of angina pectoris, and nitroglycerine consumption, and increases exercise time, and myocardial perfusion, while having no influence on arrhythmia. On the other hand, the functional class of angina pectoris increased, the exercise time decreased in patients who received only medical treatment.

In his article D. Kirklin [9] showed that the survival rate in patients with coronary artery disease without left ventricle dysfunction is about 75 % over 5 years, 60% over 10 years, and 45% over 15 years. That is why the most important indicator for the efficacy of stem cell treatment has become health-related quality of life (HRQL) [6, 7]. HRQL estimates components associated with disease and helps to understand the influence of disease and treatment of patient’s physical and mental health. It has been established that questionnaire SF-36 has good validity and reproducibility [2, 4, 15]. In our research it was shown that the functional parameters of patients have good correlation with SF-36 scales. In my opinion SF-36 can be a good guide for optimal treatment, especially in new areas such as stem cells therapy, where it is difficult to estimate reliable improvements in a patient’s condition.

Conclusions

1. Intracoronary infusion of autologous bone marrow mononuclear stem cells seems to be effective in treatment of patients who are not candidates for mechanical revascularization.

2. Questionnaire SF-36 seems to be the most effective method of estimating quality of life in patients after stem cell treatment.

3. Bodily Pain scale has strong correlation dependence with the functional class of angina pectoris (CCS).

Acknowledgements

The author wishes to thank Alexander S. Nemkov and Sergey A. Belyi (Cardio surgery Department, Saint Petersburg State Medical University n.a. Academician I.P. Pavlov, Russia) for using BMMC in clinical practice, Victor A. Krel (Cardio surgery Department, Saint Petersburg State Medical University n.a. Academician I.P. Pavlov, Russia) for intracoronary infusion of BMMC, Darya V. Ryzhkova (Science center of radiology and surgery technology, Saint Petersburg, Russia) for single-photon emission computed tomography.

References

1. Akara AR, Durdua S, Aratb M, Kilickap M, Kucuk NO, Arslan O, Kuzu I, Ozyurda U. Five-year follow-up after transepicardial implantation of autologous bone marrow mononuclear cells to ungraftable coronary territories for patients with ischaemic cardiomyopathy. Eur J Cardiothorac Surg. 2009 Oct;36(4):633-43. doi: 10.1016/j.ejcts.2009.04.045.

2. Bouchet C, Guillemin F, Paul-Dauphin A, Briançon S. Selection of quality of life measures for a prevention trial: a psychometric analysis. Control Clin Trials. 2000 Feb;21(1):30-43. doi: 10.1016/S0197-2456(99)00038-0.

3. Diederichsen AC, Moller JE, Thayssen P, Videbaek L, Saekmose SG, Barington T, Kassem M. Changes in left ventricular filling patterns after repeated injection of autologous bone marrow cells in heart failure patients. Scand Cardiovasc J. 2010 Jun;44(3):139-45. doi: 10.3109/14017430903556294.

4. Falcoz PE, Chocron S, Mercier M, Puyraveaub M, Etievent JP. Comparison of the Nottingham Health Profile and the 36-item health survey questionnaires in cardiac surgery. Ann Thorac Surg. 2002 Apr;73(4):1222-8.

6. Guyatt G, Feeny D, Patrick D. Issues in quality of life measurement in clinical trials. Control Clin Trials. 1991 Aug;12(4 Suppl):81S-90S.

7. Hawthorne G, Richardson J, Osborne R, McNeil H. The assessment of quality of life (AQoL) instrument construction, initial validation & utility scaling. Working paper (Centre for Health Program Evaluation (Australia)), 76. 2.5-6.9. Melbourne : Centre for Health Program Evaluation. 1997.

8. Hossne NA Jr, Invitti AL, Buffolo E, Azevedo S, Rodrigues de Oliveira JS, Stolf NG, Cruz LE, Sanberg PR. Refractory angina cell therapy (ReACT) involving autologous bone marrow cells in patients without left ventricular dysfunction: a possible role for monocytes. Cell Transplant. 2009;18(12):1299-310. doi: dx.doi.org/10.3727/096368909X484671.

10. Loop FD, Lytle BW, Cosgrove DM, Stewart RW, Goormastic M, Williams GW, Golding LA, Gill CC, Taylor PC, Sheldon WC. Influence of the internal-mammary-artery graft on 10-year survival and other cardiac events. N Engl J Med. 1986 Jan 2;314(1):1-6.

13. Strauer BE, Brehm M, Zeus T, Gattermann N, Hernandez A, Sorg RV, Kögler G, Wernet P. Intrakoronare, humane autologe Stammzelltransplantation zur Myokardregeneration nach Herzinfarkt. Dtsch Med Wochenschr. 2001 Aug 24;126(34-35):932-8. doi: 10.1055/s-2001-16579-2.

14. Strauer B, Yousef M, Schannwell C. The acute and long-term effects of intracoronary Stem cell Transplantation in 191 patients with chronic heARt failure: the STAR-heart study. Eur J Heart Fail. 2010 Jul;12(7):721-9.

15. Vanderzee KI, Sanderman R, Heyink J. A comparison of two multidimensional measures of health status: the Nottingham Health Profile and the Rand 36-Item Health Survey 1.0. Qual Life Res. 1996 Feb;5(1):165-74.

Introduction

Patients and Methods

Indice	BMMC group (n=17)	Control group (n=10)
Male	14 (83%)	9 (90%)
Female	3 (17%)	1 (10%)
Median age	60.2±9	62.5±7
Previous myocardial infarction	1.2±0,8	1.4±0,7
Functional class of angina pectoris (CCS)	2.9±0,4	2.3± 0,5
Exercise test (Mets)	4.6	4.6
Nitroglycerin usage (pill/week)	28	7
Arterial hypertension	12 (70%)	5 (50%)
Diabetes mellitus	2 (12%)	1(10%)
Smoking	7 (41%)	3 (30%)
Cholesterol (mmol/l)	5.5	4.6
Relapse angina pectoris after CABG	6 (35%)	4 (40%)
Distal coronary atherosclerotic damage	8 (47%)	----
Incomplete myocardial revascularization (PCI)	3 (18%)	6 (60%)
LVIDd (mm)	51.6±2	52±4
LVIDs (mm)	35.8± 2	36.5±4
LVEF (%)	57.2±4	60.3±5
One coronary vessel disease	4 (23,5%)	6 (60%)
Two coronary vessel diseases	9 (53%)	3 (30%)
Three coronary vessel diseases	4 (23.5%)	1 (10%)
Cell Ther Transplant. 2011;3:e.000097.01. doi:10.3205/ctt-2011-en-000097-table1

Results

Answers	Main group (N=17)	Control group (N=10)
Much better now	7 (35 %)	1 (10%)
Somewhat better now	8 (53 %)	-
About the same	2 (12 %)	5 (50 %)
Somewhat worse now	-	4 (40 %)
Much worse now	-	-
Cell Ther Transplant. 2011;3:e.000097.01. doi:10.3205/ctt-2011-en-000097-table2

Discussion

Conclusions

Acknowledgements

References

6. Guyatt G, Feeny D, Patrick D. Issues in quality of life measurement in clinical trials. Control Clin Trials. 1991 Aug;12(4 Suppl):81S-90S.

В исследование включено 17 пациентов с тяжелой стенокардией, не подлежащих механической реваскуляризации. Этим пациентам в период с 2008 по 2010 гг. выполняли интракоронарную инфузию аутологичных мононуклеарных стволовых клеток костного мозга. Контрольная группа насчитывала 10 пациентов. Всех пациентов обследовали до и после лечения. Перфузию миокарда оценивали с помощью однофотонной эмиссионной компьютерной томографии. Для оценки качества жизни пациентов использовали опросник SF-36. Интракоронарная инфузия аутологичных мононуклеарных стволовых клеток костного мозга способствовала уменьшению функционального класса стенокардии, потребления нитроглицерина, увеличению времени нагрузки, улучшению перфузии миокарда и не оказывала влияния на нарушения ритма. У пациентов, получавших только медикаментозную терапию, возрастал функциональный класс стенокардии и уменьшалась продолжительность физической нагрузки. Спустя год после процедуры в группе интракоронарной инфузии отмечали выраженное улучшение качества жизни. Наибольшее улучшение происходило по индексам физического функционирования, ролевого функционирования и боли. В контрольной группе существенного увеличения данных индексов не наблюдалось, наоборот, для индекса боли отмечена обратная динамика.

Ключевые слова

стволовые клетки, мононуклеарные клетки, стенокардия, болезнь сердца, аорто-коронарное шунтирование, ангиопластика, лечение стенокардии, продолжительность физической нагрузки, потребление нитроглицерина, опросник SF-36

Cardio surgery department, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

A BMMC group consisted of 17 patients with severe angina pectoris when it is impossible to use mechanical revascularization. These patients underwent autologous bone marrow mononuclear stem cells intracoronary infusion between 2008 and 2010. A control group consisted of 10 patients. All patients were examined before and after the treatment. Single-photon emission computed tomography was performed to estimate myocardial perfusion. Questionnaire SF-36 was used for an estimation of the patient’s quality of life. Intracoronary infusion of autologous bone marrow mononuclear stem cells has decreased such parameters as the functional class of angina pectoris, and nitroglycerine consumption, and has increased exercise time, and myocardial perfusion, while having no influence on arrhythmia. On the other hand, the functional class of angina pectoris increased, and the exercise time decreased in patients who received only medical treatment. At the end of one the year a great increase in quality of life in BMMC group can be observed. The best result it is seen in the Physical Functioning scale, and the Role-Physical scale, and the Bodily Pain scale. In the control group it is not seen considerably increasing in all scales and the scale Bodily Pain has changed in opposite direction.

Keywords

stem cells, mononuclear cells, angina pectoris, heart disease, coronary artery bypass surgery, angioplasty, angina treatment, exercise time, nitroglycerine consumption, questionnaire SF-36

Keywords

stem cells, mononuclear cells, angina pectoris, heart disease, coronary artery bypass surgery, angioplasty, angina treatment, exercise time, nitroglycerine consumption, questionnaire SF-36

Keywords

stem cells, mononuclear cells, angina pectoris, heart disease, coronary artery bypass surgery, angioplasty, angina treatment, exercise time, nitroglycerine consumption, questionnaire SF-36

Cardio surgery department, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

Ключевые слова

The role of CXCL12/CXCR4-mediatedchemotaxis and homing in stromal microenvironment/leukemia interaction

Animportant role in the interaction of tumor cells and stromal niches of bone marrow (BM) is played bythe chemokine CXCL12 (SDF-1, stromal cell-derived factor-1).The leukemia cells seek to infiltrate the BM's microscopic niches due to CXCL12/CXCR4–mediated chemotaxis, where stromal CXCL12 connects with CXC receptor 4 (CXCR4), located in the tumor cell membrane. As a result, tumor cells maintain self-renewal,their differentiation is blocked, and access to chemotherapeutic agents is hampered. This results in proliferation increasing and promotes survival leukemia cells. The blocking of CXCR4 stimulates the tumor cells to leave the bone marrow and to circulate in the patient's organism. As a result, the principal possibility of a new therapeutic strategy is opened on the basis of the influence ofthe CXCL12/CXCR4 system [10].

Currently, imatinib therapy is the major strategy for bcr-abl(+) CML.This therapy is based on targeted killing of the tyrosine kinase-expressing bcr-abl tumor cells. It was recently demonstrated [3] that tyrosine kinase p210BCR-ABL can inhibit CXCL12-signaling, interfering with thechemotaxis/homing process of leukemia cells. This leads to a release of immature myeloid cells from BM specific for CML and their circulation. Jin et al. [4] suggested that imatinib can act as an antagonist of bcr-abl, reconstructing CXCL12/CXCR4interaction, and it can force CML cells to migrate andconnect to the supporting BM microenvironment (Fig. 1). As result, the tumor cells are protected from the influence of therapeutic agents and this can be a potential causeof relapse. Thus, this therapeutic imatinib strategy can lead to the negative effect of indirect stromal resistance.

Figure 1. The influence of imatinib on CXCR4 expression, migration, and adhesion in the BM niche (see [4])

With the aim of verifying these hypotheses, Jin at al [4] investigated the influence of imatinib on CXCR4 expression, migration, and apoptose of leukemia cells in imatinib-sensitive and resistant CML cell lines, as well as in primary CML patient samples. They proved that imatinib blocks the influence of bcr-abl and stimulates the expression surface CXCR4 in conditions of co-cultivation of CML cells with mesenchymal stem cells (MSC). It increases the migration of CML cells to stromal cells of BM.

This proves that ITK imatinib can lead to indirect stroma resistance to therapy, associated with the CXCL12/CXCR4 system. It provides the possibility to conclude that ITK therapy should be added to any agent attenuating a CXCL12/CXCR4 interaction. Research into the role of molecular inhibitors of CXCR4 in therapy CML is currently under way [1]. Dillmann et al. [1] pointed to a potential method of influencing the SDF-1/CXCR4 mechanism by using the selective CXCR4 antagonist Plerixafor, which makes bcr-abl(+) cells more sensitive to ITK therapy in the presence of BM stromal cells. In this way it was demonstrated that destroying the indirect CXCL12/CXCR4 interaction leads to target drug effectiveness rising. These investigations exhibit the high potential for the role of CXCR4 inhibitors in CML therapy.

Lyn–dependent drug resistance caused by the microenvironment

It is well known that Lyn-kinase of the Src-kinasefamily is one of the key components of indirect CXCL12/CXCR4 migration of normal and tumor hematopoietic cells. Lyn interacts with the CXCL12/CXCR4 system and is activated by tyrosine kinase p210 bcr-abl. CXCR4 and Lyn could be placed in lipid rafts, the mobile microdomains of cell membrane, enriched by glycosphingolipids, sphingomyelin, cholesterol, and signal molecules. TKI therapy helps CXCR4 reposition into the lipid raft, where it interacts with active phosphorylated Lyn (LynTyr396) in the CML cells. Recently,a new mechanism of drug resistance was discovered. It is based on lipid raft modulation and it includes changes of multivalent CXCR4 and Lyn complex compartmentalization (Fig. 2).

It was shown that mesenchymal stem cells (MSC), which produce CXCL12, stimulate Lyn-activation in the bcr-abl(-)AML cell line HL60. This Lyn-activation in the lipid rafts of membranes is a universal mechanism, andcould explaindrug resistance for all kinds of leukemia [7]. Lyn-associated CXCR4 reposition in lipid rafts of membranes can influence the CXCL12 / CXCR4 signal path. It activates migration and enables the survival of residual leukemia cells in BM niches.

Figure 2. Mechanisms of CXCR4 localization and Lyn activation in lipid rafts (see [8]). Imatinib causes CXCR4 clustering in lipid rafts with subsequent co-localization with active p-LynTyr396. It leads to the induction of cell migration to BM stromal cells, producing CXCL12

The discovery of activated Lyn's role in the reconstitution of CXCL12/CXCR4 signal paths during bcr-abl inhibition leads to the idea that the application of Srckinase inhibitors could diminish ITK resistance, causing a microenvironment. According to this hypothesis, the double Src/abl kinase inhibitor dasatinib induces CML cell migrations to CXCL12 at a much lower level than imatinib [8]. Investigations showed that dasatinib causes an increase in the highest level of complete cytogenetic response (46% versus 28%, p<0.0001) in CML in comparison to imatinib [5]. Thus, possible dasatinib effectiveness and lower resistance to it, is caused most probably by blockade of Lyn-dependent resistance by the microenvironment. But this is not the complete effect. Therefore, the resistance to dasatinib is caused by another mechanism, which is different to the Lyn-activated one. Most probably it depends on the surface CXCR4 expressionincreasing [8].

Results of Fei et al. [2] showed that dasatinib therapy leads to CXCR4 expression increasing at the surface of cells in cases of p210 bcr-abl(+) acute lymphoblastic leukemia. In this case dasatinib and CXCR4-inhibitor combined therapy increases cell death. It demonstrates the usefulness of a TKI and CXCR4-inhibitor combination for CML and Ph (+)ALL therapy.

Results of Tabe et al. [8] demonstrate that lipid rafts of membranes provide key functional relays between Lyn and CXCR4. Thus, chemotaxis and homing of leukemia cells depends not only on CXCR4 inclusions in rafts, but depends on the cholesterol content in cell membranes. That is why statins, inhibiting synthesis of cholesterol, can change membrane properties, destroying rafts. Additionally, there are some data that simvastatin or lovastatin and alfa 2 β combination in CML cellsleads to inhibited cell growth [6, 9]. Thus cholesterol synthesis inhibition is a potential additional approach to CML therapy.

Conclusions

The current state of the researchprovides these conclusions:

1. It is necessary to investigate the role of CXCR4 inhibitors more carefully, as they have a high potential for preventing stroma-mediated drug resistance.

2. Therapy using the double Src/abl kinase inhibitor dasatinib is more effective.
This is most probably due to the influence of Lyn-kinase, which is one of the key components of indirect CXCL12/CXCR4-migration of normal and tumor hemopoietic cells.

3. It is necessary to keep in mind the potential usefulness of statin as an additional agent in CML therapy.

References

1. Dillmann F, Veldwijk MR, Laufs S, Sperandio M, Calandra G, Wenz F, Zeller J, Fruehauf S. Plerixafor inhibits chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a dose-dependent manner resulting in increased susceptibility of BCR-ABL+ cell to Imatinib and Nilotinib. Leuk Lymphoma. 2009 Oct;50(10):1676–86. doi: 10.1080/10428190903150847.

2. Fei F, Stoddart S, Müschen M, Kim YM, Groffen J, Heisterkamp N. Development of resistance to dasatinib in Bcr/Abl-positive acute lymphoblastic leukemia. Leukemia. 2010;24:813–820. doi: 10.1038/leu.2009.302.

5. Kantarjian H, Shah NP, Hochhaus A, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2010 Jun 17;362(24):2260–70.

6. Müller-Tidow C, Kiehl M, Sindermann JR, Probst M, Banger N, Zühlsdorf M, Chou TC, Berdel WE, Serve H, Koch OM. Synergistic growth inhibitory effects of interferon-alpha and lovastatin on bcr-abl positive leukemic cells. Int J Oncol. 2003;23:151–158.

8. Tabe Y, Jin L, Iwabuchi K, Wang RY, Ichikawa N, Miida T, Cortes J, Andreeff M, Konopleva M. Role of stromal microenvironment in nonpharmacological resistance of CML to imatinib through Lyn/CXCR4 interactions inlipid rafts. Leukemia. 2011 Oct 18. doi:10.1038/leu.2011.291.

9. Yang YC, Huang WF, Chuan LM, Xiao DW, Zeng YL, Zhou DA, Xu GQ, Liu W, Huang B, Hu Q. In vitro and in vivo study of cell growth inhibition of simvastatin on chronic myelogenous leukemia cells. Chemotherapy. 2008;54:438–446. doi: 10.1159/000158663.

The role of CXCL12/CXCR4-mediatedchemotaxis and homing in stromal microenvironment/leukemia interaction

Figure 1. The influence of imatinib on CXCR4 expression, migration, and adhesion in the BM niche (see [4])

Lyn–dependent drug resistance caused by the microenvironment

Conclusions

References

5. Kantarjian H, Shah NP, Hochhaus A, et al. Dasatinib versus imatinib in newly diagnosed chronic-phase chronic myeloid leukemia. N Engl J Med. 2010 Jun 17;362(24):2260–70.

В настоящее время ведутся активные исследования ключевых механизмов взаимодействия опухолевых клеток с их микроокружением. Выявление таких механизмов и разработка лекарственных средств, которые могли бы на них воздействовать, открывает новые возможности в лечении злокачественных новообразований, в том числе лейкозов. Наибольший интерес на сегодняшний день вызывают механизмы взаимодействия между лейкозными клетками и их микроокружением с участием CXCL12/CXCR4 и Lyn-киназы

Ключевые слова

микроокружение, CXCR4, иматиниб, дазатиниб, хронические миелоидный лейкоз, лекарственная резистентность, bcr-abl, Lyn

Laboratory of transplantology and molecular hematology, R.M. Gorbacheva Memorial Institute of Pediatric Hematology and Transplantation, St. Petersburg Pavlov State Medical University, St. Petersburg, Russia

There are ongoing investigations on key mechanisms of interaction between tumor cells and their microenvironment. Identification of these mechanisms and development of therapeutic agents targeting them brings up new opportunities for cancer therapy, including leukemia treatment. Today there are active discussions on mechanisms of interaction between tumor cells and their microenvironment mediated by CXCL12/CXCR4 and Lyn-kinase.

Keywords

microenvironment, CXCR4, imatinib, dasatinib, chronic myeloid leukemia, drug resistance, bcr-abl, Lyn

Keywords

microenvironment, CXCR4, imatinib, dasatinib, chronic myeloid leukemia, drug resistance, bcr-abl, Lyn

Keywords

microenvironment, CXCR4, imatinib, dasatinib, chronic myeloid leukemia, drug resistance, bcr-abl, Lyn

Ключевые слова

Introduction

Acute myeloid leukemia (AML) is a disease of malignant hematopoietic cells, which initially affect bone marrow with further suppression of its normal counterparts and infiltrate solid organs and tissues. The aims of AML patients’ treatment are the elimination of malignant cells for the further achievement of remission (complete, morphological or molecular) and the recovery of patient's bone marrow hematopoiesis. The best expected result of such treatment is the achievement of complete remission, i.e., molecular complete remission as the result of eradication of AML symptoms. The possibility of AML relapse depends upon the quality of the remission [6]. Herein, the measurement of minimal residual disease (MRD) is required for (i) the stratification of a patient’s risk and (ii) monitoring the efficiency of treatment [13].

Modern treatment regimens allow the achievement of complete morphological remission for most patients. Unfortunately, the minimal quantity of initial leukemic cells (less than 5% of nucleated cells of the bone marrow) are able to develop the relapse of AML and the further fatal outcome.

In the primary diagnosis of AML, the quantity of leukemic cells reaches up to 1010–1012 cells. After the first course of induction therapy the number of malignant cells is reduced almost 3 times, however there are millions of leukemic cells still alive. Herein the minimal residual disease is a small population of leukemic cells, which can be detected in the bone marrow provided that there are normal peripheral blood characteristics and not any extramedullar lesion focuses in patients. Thereby the MRD of AML is a persistent number of leukemic cells in bone marrow during complete remission.

Flow cytometry versus Polymerase Chain Reaction

There are two fundamental methods for the submorphological evaluation of MRD with a high sensitivity in clinical diagnostics – Polymerase Chain Reaction (PCR) and Flow Cytometry (FC). Both the PCR and FC were invented in 1980s and these methods are the gold standard of up-to-date routine diagnostics. Flow cytometry is based on the aberrant immunophenotype surface and/or intracellular characteristics of leukemic cells, whereas PCR is based on molecular or genetic aberrations of malignant cells such as (i) gene mutations, (ii) the over-expression of mRNA, (iii) fusion transcripts and so on.

The PCR method is considered to be a highly specific tool with the sensitivity of one leukemic cell in 10⁴–10⁶ of nucleated bone marrow cells, provided that the necessary technical processes are followed [8]. The main disadvantage is a limitation in using PCR in a number of patients who do not have specific genetic aberrations. According to a number of authors, half of all patients do not have molecular – specific aberrations, so this method is difficult to use for monitoring the level of MRD in such cases [11].

Multicolor flow cytometry permits the detection of one leukemic cell in 10³–10⁴ of normal nucleated bone marrow cells. The main benefit of this method is that it can be used for virtually all patients with AML (up to 95% of patients) for the monitoring of MRD in the dynamics of treatment [5], except in cases of promyelocytic leukemia where WHO strongly recommend using PCR to detect PML-RARa transcript or t(15;17).

MRD is difficult to evaluate because the development of myeloid cells occurs entirely in the bone marrow; therefore evaluation should be based on the detection of immunophenotypic characteristics, which will differ between normal and malignant bone marrow cells [12, 15]. Unfortunately, leukemia cells do not express any specific antigens that clearly distinguish them from healthy regenerating bone marrow cells. However, some authors report that malignant cells have a fixed aberrant expression of myeloid inhibitory c-type lectin (hMICL) and CD123 and this could be used as the immunophenotypic marker for an assessment of MRD [14].

Leukemia-associated immunophenotype of AML

There are some recommendations for the initial diagnosis of AML [4]. In the primary diagnosis of immunophenotype leukemic cells, the application of combination multicolor monoclonal antibodies can be used later in the detection of aberrations in MRD [17, 16]. The leukemia-associated immunophenotype (LAIP) of the malignant cells is detected individually for a patient. The aberration detection of surface immunophenotype can be performed as (i) an asynchronous expression in one or more antigens, (ii) a cross-lineage expression in one or more antigens, (ii) a lack of expression in one or more antigens, and (iv) an over-expression in one or more markers. The precise evaluation of MRD in patients with AML with multicolor FC depends upon many different factors including the strategy of gating (using the CD45/SS or FS/SS gating strategies) [9], a broad panel of monoclonal antibodies, instrument settings (a compensation), and the operator’s qualification (Fig. 1, Fig. 2).

Some complications arise at the diagnosis of AML during the detection of LAIP. AML in comparison with ALL is rather heterogeneous disease, and very often it produces two or more subpopulations. Some authors very actively propose using a special strategy for the measurement of LAIP. On the other hand, there are some questions such as whether we should evaluate the subpopulation of “leukemic stem” cells CD34+CD38- at diagnosis even if it appears at very low frequency (for instance, in the case of promyelocytic leukemia) and should we take into account the changes of granularity according to SS parameter in CD45/SS gating strategy at the diagnosis and during the follow-up of patients.

Another issue is stability of the LAIPs during treatment and at the relapse of AML. This question merits additional discussion. Briefly, in concordance with a number of authors the best way to reveal alterations in LAIPs is to use a broad panel of monoclonal antibody at the primary diagnosis of AML. It increases the probability of detecting LAIPs during follow-up.

Figure 1. Detection of a LAIP at the diagnosis of AML using multicolor flow cytometry and CD45-SS gating strategies – CD45dimCD4+CD13+CD33+CD34+CD38+CD117+ with the co-expression of CD7 and CD64. The type of aberrant expression is asynchronous expression of CD4 and co-expression CD64 with early myeloid antigens

Figure 2. After induction chemotherapy and leukocyte recovery, persistent cells with LAIP are detectable at a very low frequency

Conclusion

There are different options to detect MRD for patients with AML. In patients with detectable molecular markers, both PCR and multicolor FC might be used for receiving more sensitive and specific information about MRD. The leukemic cells are detectable by FC with sensitivity lower than molecular aberrations by PCR, however some patients do not have such mutations. One of the main problems is standardizing procedures in diagnostic AML by both PCR and FC. There is no official protocol with comparisons between PCR and FC during a follow-up patient with AML.

The identification of LAIPs in patients with AML depends upon (i) how broad the panel of reagents is used at diagnosis and during an evaluation of MRD, (ii) how appropriate settings are used in flow cytometer (compensation, laser stability, voltage) and (iii) the operator’s qualifications [1].

The search for new markers to distinguish normal from malignant cells after chemotherapy in bone marrow by multicolor FC is a future direction of development in clinical laboratory diagnostics, and the possibility to identify new target treatment with AML patients [7].

Acknowledgements

The author wish to thank Yekaterina Ye. Zueva (Center for laboratory diagnostics, Pavlov Medical University of St. Petersburg, Russia) for providing the supervision.

References

4. Campana D. Progress of minimal residual disease studies in childhood acute leukemia. Curr Hematol Malig Rep. 2010 Jul;5(3):169-7. doi: 10.1007/s11899-010-0056-8.

5. Cheson BD, Bennett JM, Kopecky KJ, Büchner T, Willman CL, Estey EH, Schiffer CA, Doehner H, Tallman MS, Lister TA, Lo-Coco F, Willemze R, Biondi A, Hiddemann W, Larson RA, Löwenberg B, Sanz MA, Head DR, Ohno R, Bloomfield CD; International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. Revised Recommendations of the International Working Group for Diagnosis, Standardization of Response Criteria, Treatment Outcomes, and Reporting Standards for Therapeutic Trials in Acute Myeloid Leukemia. Journal of Clinical Oncology. 2003;21(24):4642-9.

6. Chung NG, Buxhofer-Ausch V, Radich JP. The detection and significance of minimal residual disease in acute and chronic leukemia. Tissue Antigens. 2006 Nov;68(5):371-85. doi: 10.1111/j.1399-0039.2006.00714.x.

9. Kern W, Voskova D, Schnittger S, Schoch C, Hiddemann W, Haferlach T. Four-fold staining including CD45 gating improves the sensitivity of multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia. Hematol J. 2004;5(5):410-8.

11. Kern W, Bacher U, Haferlach C, Schnittger S, Haferlach T. The role of multiparameter flow cytometry for disease monitoring in AML. Best Pract Res Clin Haematol. 2010 Sep;23(3):379-90. doi: 10.1016/j.beha.2010.06.007.

12. Kern W, Schoch C, Haferlach T, Schnittger S. Monitoring of minimal residual disease in acute myeloid leukemia. Crit Rev Oncol Hematol. 2005 Nov;56(2):283-309. doi: 10.1016/j.critrevonc.2004.06.004.

14. Sperr WR, Valent P. Novel developments in AML – ASH 2010. ASH report. Memo 2011;4:106-9.

15. Voskova D, Schnittger S, Schoch C, Haferlach T, Kern W. Use of five-color staining improves the sensitivity of multiparameter flow cytomeric assessment of minimal residual disease in patients with acute myeloid leukemia. Leuk Lymphoma 2007;48(1):80-8. doi: 10.1080/10428190600886164.

Introduction

Flow cytometry versus Polymerase Chain Reaction

Leukemia-associated immunophenotype of AML

Figure 2. After induction chemotherapy and leukocyte recovery, persistent cells with LAIP are detectable at a very low frequency

Conclusion

Acknowledgements

The author wish to thank Yekaterina Ye. Zueva (Center for laboratory diagnostics, Pavlov Medical University of St. Petersburg, Russia) for providing the supervision.

References

4. Campana D. Progress of minimal residual disease studies in childhood acute leukemia. Curr Hematol Malig Rep. 2010 Jul;5(3):169-7. doi: 10.1007/s11899-010-0056-8.

14. Sperr WR, Valent P. Novel developments in AML – ASH 2010. ASH report. Memo 2011;4:106-9.

Минимальная остаточная болезнь (МОД) у больных с диагнозом острого миелобластного лейкоза (ОМЛ) может выявляться различными методами. Знание уровней МОД весьма важно для (1) стратификации больных по группамриска, и (2) для мониторинга эффективности лечения. Разработаны процедуры проточной цитометрии для идентификации поверхностных и внутриклеточных антигенов в клетках. Они стали исключительно полезным способом определения клеток костного мозга, с иммунофенотипом, ассоциированным с лейкозом, и они осществимы практически у всех больных с ОМЛ. Данное краткое сообщение касается, в основном, оценки МОД при ОМЛ с помощью многоцветной проточной цитометрии.

Ключевые слова

острый миелобластный лейкоз, многоцветная проточная цитометрия, иммунофенотип, ассоциация с лейкозом

Center for laboratory diagnostics, Pavlov State Medical University, St. Petersburg, Russia

Correspondence
Konstantin U. Slobodnyuk, Center for laboratory diagnostics, Pavlov State Medical University, 6/8 Lev Tolstoy Str., St. Petersburg, 199022, Russia
E-mail: k.slobodnyuk@gmail.com

Minimal residual disease (MRD) in patients diagnosed with acute myeloid leukemia (AML) can be detected by different methods. Knowing the MRD value is highly important for (i) stratification of patients in to risk groups and (ii) monitoring treatment's efficiency. Flow cytometry procedures have been developed to identify surface and intracellular antigens in cells. It has become an extremely useful tool for detecting the leukemia-associated immunophenotype of leukemia cells in bone marrow and it can be provided to virtually all patients with acute myeloid leukemia. This short report focuses on the assessment of AML MRD with multicolor flow cytometry.

Keywords

acute myeloid leukemia, multicolor flow cytometry, leukemia-associated immunophenotype

Keywords

acute myeloid leukemia, multicolor flow cytometry, leukemia-associated immunophenotype

Keywords

acute myeloid leukemia, multicolor flow cytometry, leukemia-associated immunophenotype

Center for laboratory diagnostics, Pavlov State Medical University, St. Petersburg, Russia

Correspondence
Konstantin U. Slobodnyuk, Center for laboratory diagnostics, Pavlov State Medical University, 6/8 Lev Tolstoy Str., St. Petersburg, 199022, Russia
E-mail: k.slobodnyuk@gmail.com

Center for laboratory diagnostics, Pavlov State Medical University, St. Petersburg, Russia

Correspondence
Konstantin U. Slobodnyuk, Center for laboratory diagnostics, Pavlov State Medical University, 6/8 Lev Tolstoy Str., St. Petersburg, 199022, Russia
E-mail: k.slobodnyuk@gmail.com

Ключевые слова

острый миелобластный лейкоз, многоцветная проточная цитометрия, иммунофенотип, ассоциация с лейкозом

Ключевые слова

острый миелобластный лейкоз, многоцветная проточная цитометрия, иммунофенотип, ассоциация с лейкозом