Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro
To test the ability of cultured mesenchymal stem cells (MSCs) from bone marrow (BM) of children with oncohematological diseases after chemotherapy and radiation to support proliferation and self renewal of hematopoietic cells in vitro. Methods: BM samples of 8 patients and 9 healthy children-donors...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2008
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| Назва видання: | Experimental Oncology |
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| Цитувати: | Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro / Ya. Isaikina, T. Shman // Experimental Oncology. — 2008. — Т. 30, № 2. — С. 121–128. — Бібліогр.: 29 назв. — англ. |
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irk-123456789-1391952018-06-20T03:12:50Z Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro Isaikina, Ya. Shman, T. Uncategorized To test the ability of cultured mesenchymal stem cells (MSCs) from bone marrow (BM) of children with oncohematological diseases after chemotherapy and radiation to support proliferation and self renewal of hematopoietic cells in vitro. Methods: BM samples of 8 patients and 9 healthy children-donors used for MSCs preparation applying techinque of expansion in vitro. CD34+ cells were isolated from BM of donors. The ability of MSCs to maintain hematopoietic stem cells (HSCs) proliferation was tested in semisolid methylcellulose medium and in liquid long-term-culture (LTC) medium. Results: The presence of MSCs derived from BM of patients in methylcellulose medium induced 2-fold increase of the number of commited myeloid progenitors without cytokines, 7-fold increase together with growth factors and 14-fold increase of the amount of earlier pluripotent hematopoietic precursor cells (CFU-GEMM) compared to expansion of HSCs without MSCs and cytokines. The presence of MSCs layer of patients in liquid LTC medium significantly promoted the hematopoietic cells proliferation rate, measured on 7th, 14th and 21st day. The total number of cells was multiplied 161.2-fold on 21st day as compared to 116.4-fold without MSCs layer. In the presence of MSCs layer, we detected the increase of proportion of bipotent CFU-GM precursors from 4% to 11% and pluripotent CFU-GEMM precursors from 0.1% to 0.6% in population of HSCs. In both types of experiments the capacity of patients’ MSCs to support HSCs proliferation and self renewal was the same as for healthy donors’ MSCs. Conclusion: In this study, МSCs were isolated from BM of children with malignancies after high-dose chemotherapy or radiation. The ability of these MSCs to maintain hematopoiesis in vitro was tested. It was shown that co-transplantation of autologous MSCs is a good way to improve hematopoietic stem cells engraftment and reduce a period of granulocytopenia after autologous HSCs transplantation in case of insufficient CD34+ cell number in autologous transplant. Цель: исследовали способность культивируемых in vitro мезенхимальных стволовых клеток (МСК), выделенных из костного мозга детей со злокачественными новообразованиями, получавших в качестве лечения высокодозовую полихимиотерапию, поддерживать гемопоэз in vitro. Материалы и методы: МСК выделяли и наращивали из проб костного мозга 8 пациентов и 9 здоровых доноров детского возраста. CD34+ клетки выделяли из проб костного мозга доноров. Были поставлены эксперименты по совместному культивированию МСК и CD34+ клеток в среде метилцеллюлозы и в среде для долгосрочного культивирования с добавлением цитокинов и при их отсутствии. Результаты: в метилцеллюлозной среде наличие МСК пациентов увеличивало количество коммитированных миелоидных предшественников в 2 раза, МСК совместно с ростовыми факторами стимулировало пролиферацию этого типа клеток в 7 раз, а полипотентных предшественников — в 14 раз. При долгосрочном культивировании с цитокинами на слое МСК общее количество ГСК на 21-й день возростало в 161,2 раза. При пролиферации CD34+ клеток в среде с МСК содержание бипотентных КОЕ-ГМ повысилось с 4 до 11%, а полипотентных КОЕ-ГЭММ — с 0,1 до 0,6%. В экспериментах по экспансии CD34+ клеток результаты при использовании в качестве фидерного слоя МСК пациентов или МСК доноров статистически не отличались. Выводы: проведенные исследования позволяют сделать вывод о том, что МСК из костного мозга пациентов со злокачественными новообразованиями обладают достаточным функциональным потенциалом для пролиферации и самоподдержания ГСК. Ко-трансплантация аутологичных МСК при аутотрансплантации ГСК с низким содержанием CD34+ клеток в трансплантате может являться перспективной для сокращения периода восстановления гемопоэза в ранний посттрансплантационный период. 2008 Article Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro / Ya. Isaikina, T. Shman // Experimental Oncology. — 2008. — Т. 30, № 2. — С. 121–128. — Бібліогр.: 29 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/139195 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Uncategorized Uncategorized |
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Uncategorized Uncategorized Isaikina, Ya. Shman, T. Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro Experimental Oncology |
| description |
To test the ability of cultured mesenchymal stem cells (MSCs) from bone marrow (BM) of children with oncohematological diseases after chemotherapy and radiation to support proliferation and self renewal of hematopoietic cells in vitro. Methods: BM samples of 8 patients and 9 healthy children-donors used for MSCs preparation applying techinque of expansion in vitro. CD34+ cells were isolated from BM of donors. The ability of MSCs to maintain hematopoietic stem cells (HSCs) proliferation was tested in semisolid methylcellulose medium and in liquid long-term-culture (LTC) medium. Results: The presence of MSCs derived from BM of patients in methylcellulose medium induced 2-fold increase of the number of commited myeloid progenitors without cytokines, 7-fold increase together with growth factors and 14-fold increase of the amount of earlier pluripotent hematopoietic precursor cells (CFU-GEMM) compared to expansion of HSCs without MSCs and cytokines. The presence of MSCs layer of patients in liquid LTC medium significantly promoted the hematopoietic cells proliferation rate, measured on 7th, 14th and 21st day. The total number of cells was multiplied 161.2-fold on 21st day as compared to 116.4-fold without MSCs layer. In the presence of MSCs layer, we detected the increase of proportion of bipotent CFU-GM precursors from 4% to 11% and pluripotent CFU-GEMM precursors from 0.1% to 0.6% in population of HSCs. In both types of experiments the capacity of patients’ MSCs to support HSCs proliferation and self renewal was the same as for healthy donors’ MSCs. Conclusion: In this study, МSCs were isolated from BM of children with malignancies after high-dose chemotherapy or radiation. The ability of these MSCs to maintain hematopoiesis in vitro was tested. It was shown that co-transplantation of autologous MSCs is a good way to improve hematopoietic stem cells engraftment and reduce a period of granulocytopenia after autologous HSCs transplantation in case of insufficient CD34+ cell number in autologous transplant. |
| format |
Article |
| author |
Isaikina, Ya. Shman, T. |
| author_facet |
Isaikina, Ya. Shman, T. |
| author_sort |
Isaikina, Ya. |
| title |
Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro |
| title_short |
Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro |
| title_full |
Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro |
| title_fullStr |
Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro |
| title_full_unstemmed |
Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro |
| title_sort |
influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2008 |
| topic_facet |
Uncategorized |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/139195 |
| citation_txt |
Influence of mesenchymal stem cells derived from bone marrow of children with oncohematological diseases on proliferation and self-renewal of hematopoietic progenitor cells in vitro / Ya. Isaikina, T. Shman // Experimental Oncology. — 2008. — Т. 30, № 2. — С. 121–128. — Бібліогр.: 29 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
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| first_indexed |
2025-07-10T07:47:23Z |
| last_indexed |
2025-07-10T07:47:23Z |
| _version_ |
1837245403537342464 |
| fulltext |
Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ���
High-dose chemotherapy alone and sometimes
together with radiotherapy with following a�tologo�s
transplantation of hematopoietic stem cells �HSCs� is
�sed for treatment of patients with advanced stages
or relapses of malignant diseases. However� recon-
stit�tion of ne�trophil and platelet cell n�mber after
a�totransplantation of HSCs is delayed for abo�t 5%
of patients. In most cases it co�ld be explained by low
dose of transplanted CD�4+ cells per kg of patient’s
body weight [�� �].
Dimitri et al observed that the n�mber and q�ality
of mobilized peripheral blood stem cells �PBSC� was
low in patients that received m�ltiple ro�nds of che-
motherapy and grafts with low n�mbers of HSCs. Poor
q�ality of HSCs was the ca�se of graft fail�re �pon their
a�tologo�s inf�sion [�].
Recently� some approaches were s�ggested
to solve this problem. One of them is a generally
accepted proced�re of repeated attempts of PBSC
collection or aspiration of considerable vol�me
of bone marrow �BM� in case of ins�fficient CD�4+ cell
n�mber in transplant at the time of primary collection
of PBSC. Another approach incl�des expansion of
hematopoietic stem cells ex vivo. This is an attractive
method to improve hematological recovery or red�ce
graft size. However� altho�gh clinical trials indicated
that total n�mber of CD�4+ cells and primitive stem
cells in PBSC can be s�ccessf�lly expanded in vitro�
hematopoietic recovery by �sing s�ch grafts is not im-
proved [4� 5]. This co�ld be explained by the red�ction
of stem cells ability to home to the bone marrow after
inc�bations with several cytokine combinations� which
leads to the loss of their ability to repop�late the he-
matopoietic system of irradiated recipients [6].
At the present time� there is another highly attrac-
tive approach to red�ce the hematopoiesis reconstit�-
tion period after transplantation of HSCs — the sim�l-
taneo�s a�totransplantation of mesenchymal stem
cells �MSCs�. Mesenchymal stem cells are prec�rsors
of stromal stem cells� osteoblasts� adipocyts� en-
dothelial cells� which form regions of hematopoietic
ind�ctive environment in bone marrow� th�s� s�pport-
ing the prod�ction of le�cocytes� red cells and platelets
[7� �]. From one hand� their impact on hematopoiesis
prec�rsors is realized by secretion of sol�ble cyto-
kines� chemokines� peptides� mediators and hor-
mones. From the other hand� MCSs form the extra-
INFLUENCE OF MESENCHYMAL STEM CELLS DERIVED FROM
BONE MARROW OF CHILDREN WITH ONCOHEMATOLOGICAL
DISEASES ON PROLIFERATION AND SELF-RENEWAL
OF HEMATOPOIETIC PROGENITOR CELLS IN VITRO
Ya. Isaikina*, T. Shman
Belarusian Research Centre for Pediatric Oncology and Hematology, Minsk, Belarus
Aim: To test the ability of cultured mesenchymal stem cells (MSCs) from bone marrow (BM) of children with oncohematological disea
ses after chemotherapy and radiation to support proliferation and self renewal of hematopoietic cells in vitro. Methods: BM samples
of 8 patients and 9 healthy childrendonors used for MSCs preparation applying techinque of expansion in vitro. CD34+ cells were
isolated from BM of donors. The ability of MSCs to maintain hematopoietic stem cells (HSCs) proliferation was tested in semisolid
methylcellulose medium and in liquid longtermculture (LTC) medium. Results: The presence of MSCs derived from BM of patients
in methylcellulose medium induced 2fold increase of the number of commited myeloid progenitors without cytokines, 7fold increase
together with growth factors and 14fold increase of the amount of earlier pluripotent hematopoietic precursor cells (CFUGEMM)
compared to expansion of HSCs without MSCs and cytokines. The presence of MSCs layer of patients in liquid LTC medium sig
nificantly promoted the hematopoietic cells proliferation rate, measured on 7th, 14th and 21st day. The total number of cells was
multiplied 161.2fold on 21st day as compared to 116.4fold without MSCs layer. In the presence of MSCs layer, we detected the
increase of proportion of bipotent CFUGM precursors from 4% to 11% and pluripotent CFUGEMM precursors from 0.1% to 0.6%
in population of HSCs. In both types of experiments the capacity of patients’ MSCs to support HSCs proliferation and self renewal
was the same as for healthy donors’ MSCs. Conclusion: In this study, МSCs were isolated from BM of children with malignancies
after highdose chemotherapy or radiation. The ability of these MSCs to maintain hematopoiesis in vitro was tested. It was shown
that cotransplantation of autologous MSCs is a good way to improve hematopoietic stem cells engraftment and reduce a period of
granulocytopenia after autologous HSCs transplantation in case of insufficient CD34+ cell number in autologous transplant.
Key Words: mesenchymal stem cells, oncohematological diseases, hematopoiesis, hematopoietic progenitors.
Received: February 5, 2008.
*Correspondance: Fax: 265-42-22
E-mail: yaninai@mail.ru
Abbreviations used: BFU-E — burst-forming units erythroid; BM —
bone marrow; GF — growth factors; CFU-G — colony forming
units — granulocyte; CFU-GEMM — colony forming units — granu-
locyte-erythroid-monocyte-megakaryocyte; CFU-M — colony form-
ing units — macrophage; CFU-GM — colony forming units — granu-
locyte and macrophage; HBSS — Hanks Balanced Salt Solution;
HSCs — hemopoietic stem cells; IL-3 — interleukin-3; IMDM —
Iscove Modified Dulbecco Medium; LTC-IC — long-term-culture
initiating cell; MNCs — mononuclear cells; МSСs — mesenchymal
stem cells; PBC — peripheral blood stem cells; rhGM-CSF — re-
combinant human granulocyte-macrophage colony-stimulating
factor; rhSCF — recombinant human stem cell factor; SCT — stem
cells transplantation; HPC — hematopoietic progenitor cell.
Exp Oncol ����
��� �� �������
��� Experimental Oncology ��� �������� ���� ���ne�
cell�lar matrix from collagen� fibronectin and laminin
molec�les� which provide adhesion of hematopoietic
cells. Stromal cells contin�o�sly synthesize colony
stim�lating factor �CSF�� interle�kin-6 �IL-6�� IL-��
IL-7� IL-�� SCF� Flt-�-ligand� GM-CSF� thrombopoietin�
ins�lin-like growth factor� transforming growth factor
�TGF�� th�s� s�pporting the maintenance of the de-
termined level of blood cells n�mber. IL-� is the main
cytokine prod�ction ind�ctor� and TGF is able to inhibit
hematopoiesis [9]. The ability of MSCs to s�pport he-
matopoiesis in vitro has been shown by many a�thors
in sim�ltaneo�s c�ltivation of MSCs and HSCs [��].
The capacity of stromal cells to s�pport the expansion
of primitive hematopoietic progenitors for more than
�� weeks was revealed [��].
Until recently� practically all of the investigations of
MSCs effect on hematopoietic prec�rsor cells prolife-
ration and self-renewal were carried o�t by �sing
stromal cells layer derived from bone marrow MSCs
of healthy donors. Therefore� it is important to st�dy
f�nctional potential of mesenchymal stem cells derived
from bone marrow of children� who are the candidates
for HSCs transplantation after high dose chemo-
therapy. We investigated the ability of c�lt�red MSCs
from patients’ BM to s�pport proliferation and self-
renewal of hematopoietic cells in vitro. It can be of
great importance for creation of therape�tical strategy
of a�tologo�s MSCs application for hematopoiesis
s�pport and ne�tropenia period red�ction after HSCs
a�tologo�s transplantation for children with ins�f-
ficiency of CD�4+ cell n�mber in graft.
MATERIALS AND METHODS
Materials. Bone marrow samples of � patients
with IV stage of non-Hodgkin’s lymphoma� Hodgkin
disease and Ewing sarcoma have been �sed. Protocol
of treatment of these patients incl�ded a�tologo�s
transplantation of HSCs. Comparative analysis has
been performed by eval�ation of bone marrow sam-
ples of 9 children-donors of hemopoietic stem cells
for allogenic transplantation.*
BM cell preparation. Samples of BM were obtained
from allogenic or a�tologo�s bone marrow transplants.
Monon�clear cells �MNCs� were isolated by centrif�ga-
tion on a Ficoll-Hypaq�e gradient �density �.�77 g/ml�
�Sigma� USA� at 4�� g for �5 min� washed twice in Hanks
Balanced Sold Sol�tion �HBSS� �Sigma� USA�.
CD34+ cells separation. CD�4+ cells were derived
from bone marrow of healthy donors. The proced�re
of CD�4+ cells processing from fraction of bone marrow
monon�clear cells was carried by means of H�man CD�4
Selection Kit for positive selection �StemCell Technolo-
gies� Canada� according to man�fact�rer’s protocol.
Human mesenchymal cells preparation. Mono-
n�clear cells were s�spended in Iscove Modified D�l-
becco Medi�m �IMDM� with ��% fetal bovine ser�m
�FBS� �Sigma� USA� at concentration of � х ��6/ml
and were transferred to tiss�e c�lt�re flask �Sarstedt�
* Patients provided written consent to perform the study, and research
was approved by the Ethic Committee of the Research Center.
Germany� at density �.5 х ��6/cm�. Cells were c�lt�red
at �7 C with 5% CO�� in h�midified atmosphere. When
cells achieved ���9�% of confl�ence� the adherent
layer was washed with HBSS to remove resid�al FBS
and was detached with �.�5% trypsin-EDTA sol�tion
�Sigma� USA�. Then MSCs were washed in IMDM
with ��% fetal bovine ser�m and �.6�� х ��6 cells were
transferred to a new �5 sm2 flask �I passage�. When
~ 9�% confl�ent layer was prod�ced� the manip�lation
was repeated again. For eval�ation of mesenchymal
cells infl�ence on hematopoietic cell growth we �sed
the same MSCs from II�IV passages for the healthy
donors’ and patients’ preparations.
Long-term culture hematopoietic and mesen-
chymal stem cells. Mesenchymal cells were seeded
at concentration of �.5 х ��5/well into �4-wells plates.
Cells were c�lt�red in IMDM-medi�m s�pplemented
with ��% FBS overnight at �7 °C in 5% CO�. Monolayer
of cells was treated with �% gl�taraldehyde or irradia-
ted at �� Gy to stop proliferation of mesenchymal cells.
Then� p�rified donors’ CD�4+ cells �� x ��4/ml� were
added to the wells. Cells were c�lt�red in IMDM s�pple-
mented with �5% FBS� � mM L-gl�tamine and �-mer-
captoethanol in presence and witho�t a cocktail of re-
combinant h�man cytokines. We �sed following growth
factors �GF�: IL-� ��� ng/ml�� IL-6 ��� U/ml�� stem cell
factor SCF �5� ng/ml�� Flt-�-ligand ���� ng/ml�. Cells
were inc�bated for �� days in standard conditions with
weekly replacement of half of the medi�m. Cells were
co�nted and their viability was analyzed at 7th� �4th
and ��st day.
Semisolid assay of clonogenic progenitors
in the different conditions. Pl�ripotent and line-
committed hematopoietic prec�rsor cells form dif-
ferent types of colonies or «colony-forming �nits»
�CFU� in short-term c�lt�re. Myeloid line-committed
prec�rsors form colony-forming �nits gran�locyte
�CFU-G�� CFU — macrophage �CFU-M�; bypotential
prec�rsor forms CFU — gran�locyte-macrophage
�CFU-GM�; pl�ripotential hematopoietic prec�rsor
forms CFU- gran�locyte — erythroid-monocyte-
megakaryocyte �CFU-GEMM� and erythroid prec�rsor
forms b�rst-forming �nits erythroid �BFU-E�. Experi-
ments were performed in six parallel variants. Initially�
CD�4+ cells were s�spended at a concentration � x
��4 cells/ml in � ml complete methylcell�lose me-
di�m ��% methylcell�lose in IMDM� ��% fetal bovine
ser�m��% alb�min� � mM L-gl�tamine� witho�t and
with presence of colonystim�lating growth factors:
�� ng/mL recombinant h�man IL-� �rhIL-��; �� ng/mL
recombinant h�man gran�locyte macrophage — CSF
�rhGM-CSF�; 5� ng/mL recombinant h�man stem cell
factor �rhSCF�; � U/mL h�man erythropoietin �Epo�.
F�rther� each of the two variants of cells in methylcel-
l�lose medi�m were plated �� x ��4 cells/well in � ml�
in presence and witho�t MSC layer derived from bone
marrow of patients and from bone marrow of donors
as a control in 6 wells of �4-wells plastic microplates.
Plates were inc�bated at �7 °C with 5% C�� and ≥ 95%
h�midity for �� days. N�mber of CFU-G� CFU-M�
Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ���
CFU-GM� CFU-GEMM and BFU-E were scored mac-
roscopically at ��st day.
Statistical analysis. Statistical processing of
data was performed with the statistical package
STATISTICA � Ver 6.�. �Stat Soft Inc� USA�. Mann —
Whitney U test was �sed to eval�ate significant diffe-
rences between samples of patients and donors. Wil-
coxon matched pairs test was �sed to eval�ate the fold
increase of cells c�lt�red �nder different condition.
RESULTS
Effect of MSCs on maintaining of CD34+ cells
proliferation in semisolid medium. Research was
performed �sing MSCs derived from bone marrow
of � patients and 9 healthy donors and hematopoi-
etic CD�4+ cells selected from samples of healthy
donors. Eval�ation of clonogenic progenitors content
was carried o�t after c�ltivation of CD�4+ cells in
methylcell�lose medi�m for �� days in each of 6 vari-
ants: hematopoietic cells as the negative control� he-
matopoietic cells with MSCs of donors� hematopoietic
cells with MSCs of patients� hematopoietic cells with
growth factors �GF�� hematopoietic cells with GF and
MSCs of donors� and hematopoietic cells with GF and
MSCs of patients. All types of myeloid line colonies�
i. e. CFU-G� CFU-M� CFU-GM were termed “myeloid
progenitors”. The level of variability in myeloid pro-
genitor cells n�mber �nder different c�lt�ral condi-
tions was relative to myeloid progenitor’s n�mber in
negative control �Fig. �� a�. Analysis of res�lts showed
relative increase in myeloid progenitors n�mber when
CD�4+ cells were c�lt�red in the presence of GF
alone� MSCs alone or GF and MSCs sim�ltaneo�sly.
The presence of both patients’ and donors’ MSCs in
methylcell�lose medi�m res�lted in �.�6 ± �.�5-fold
�p < �.�5� and in �.�5 ± �.� �p < �.�5� — fold increase
in the n�mber of myeloid progenitors cells respectively
as compared with negative control� while adding only
growth factors in methylcell�lose medi�m increased
CFU-GM n�mber �.� ± �.6�-fold. Hematopoietic stem
cells proliferated more intensively in the sim�ltaneo�s
presence of GF and MSCs in growth medi�m. Myeloid
progenitors n�mber on average was 7.�� ± �.�4-fold
higher in the presence of patients’ MSCs �p < �.���
and 7.�� ± �.�5-fold higher in the presence of do-
nors’ MSCs as compared with the negative control
�p < �.���. The significant difference between impacts
of patients and donors’ MSCs on proliferation of the
myeloid prec�rsor cells was not detected.
Infl�ence of both patients’ and donors’ MSCs
on the earlier pl�ripotent hematopoietic prec�rsor
cells proliferation was eval�ated CFU-GEMM n�mber
in each of 6 variants. The Fig. �� b presents relative
increase in CFU-GEMM n�mber in case of GF alone or
GF and MSCs sim�ltaneo�sly added with CD�4+cells.
We didn’t observe any CFU-GEMM n�mber expan-
sion when CD�4+ cells were c�lt�red in the presence
of MSC witho�t GF. Maxim�m increase in CFU-GEMM
n�mber was detected when GF and MSCs were added
sim�ltaneo�sly as compared with variants of MSCs
alone ��5.9 ± 7.� vs �.9 ± �.4 for donors �p < �.�5�
and �4.�7 ± 6.9 vs �.9 ± �.� for patients �p < �.�5��
or GF alone ��5.9 ± 5.9 vs �.� ± �.� from donors
�p < �.�5� and �4.�7 ± 6.9 vs 5.�� ± �.�6 from patients
�p < �.�5��.
Fig. 1. Effect of MSC derived from BM of patients with oncohe-
matological diseases on proliferation of CD�4+ cells in semisolid
medi�m. MSCs derived from donors’ BM served as a control.
� x ��4 of CD�4+ cells of healthy donors were c�lt�red in methyl-
cell�lose medi�m �nder different conditions: c�lt�re medi�m
alone; with GF; with patients’ MSCs; with GF and patients’ MSCs;
with donors’ MSCs; with GF and donors’ MSCs. Cells were c�l-
t�red for �� days and n�mber of CFU was scored. Each col�mn
represents the fold increase of progenitors observed in six sepa-
rate variants� relative to n�mber of progenitors grown witho�t
GF and MSCs: (a) Fold increase of total n�mber of CFU-G +
CFU-M + CFU-GM and termed here as “myeloid progenitors”;
(b) Fold increase of pl�ripotential prec�rsors cells �CFU-GEMM�.
Wilcoxon matched pairs test was �sed for comparison of fold
cells increase inside each gro�p �*p < �.�5� **p < �.���
Mann — Whitney U test was �sed to compare the
effect of donors’ and patients’ MSCs on fold increase
of hematopoietic cells’ n�mbers. It confirmed that
MSCs derived from patients’ BM with statistical validi-
ty had the same s�pporting effect on the proliferation
of the pl�ripotent hematopoietic prec�rsor cells �CFU-
GEMM� as donors’ MSCs.
Influence of MSCs on maintaining of CD34+
cells proliferation in liquid long-term cell culture.
In order to investigate the infl�ence of MSCs on he-
matopoietic cells expansion in liq�id complete growth
medi�m� two experiments with fo�r c�ltivation variants
were performed: �� �n = �� — only CD�4+ cells� CD�4+
cells on MSCs layer of healthy donors� CD�4+ cells
with growth factors� CD�4+ cells with growth factors on
MSCs layer of healthy donors; �� �n = �� — only CD�4+
cells� CD�4+ cells on MSCs layer of patients� CD�4+ cells
with GF� CD�4+ cells with GF on MSCs layer of patients.
For both experiments� CD�4+ cells were isolated from
��4 Experimental Oncology ��� �������� ���� ���ne�
bone marrow of healthy donors’ samples� and the ini-
tial concentrations of these cells were � х ��4 for each
c�ltivation variants. Kinetics of СD�4+ cells proliferation
for different variants is presented on Fig. �. The absol�te
cells n�mbers from � variants of both experiments� when
MSCs layer was absent and CD�4+cells were grown only
in c�lt�re medi�m� or CD�4+ cells were grown in the
presence of GF alone� were integrated �n = �6 in every
case�. Res�lts represented the mean absol�te cells
n�mber ± SD of six different experiments on 7th� �4th�
and ��st day of c�lt�ring. We did not observe significant
difference in cells n�mber when СD�4+ cells were c�lti-
vated in ser�m-containing c�lt�re medi�m� and the total
amo�nt of cells was �.� ± �.�7 x ��4� �.55 ± �.�� x ��4
and �.5 ± �.�� x ��4 on 7th� �4th and ��st day� respec-
tively. The presence of both donors’ and patients’ MSCs
layer witho�t GF had no effect on significant differences
of absol�te n�mber of growing cells d�ring �� days. Total
amo�nt of cells in the presence of donors MSCs was
�.5 ± �.6 x ��4� �.� ± �.� x ��4 and �.� ± �.� x ��4 on 7th�
�4th and ��st day� respectively� and in the presence of
patients’ MSCs was �.� ± �.7 x ��4� �.75 ± �.7 x ��4 and
�.6 ± �.� x ��4 on 7th� �4th and ��st day� respectively. The
presence of cytokines in c�lt�re medi�m significantly
increased the absol�te n�mber of cells from � x ��4 to
�5.5 ± �.5 x ��4� to 5� ± ��.� x ��4 and to ���.5 ± ��.� x ��4
on day 7� �4 and �� respectively� p < �.�5. The increase
of total cells n�mber when CD�4+ cells were c�lt�red
in the presence of both donors’ and patients’ MSCs
and cytokines was the most evident� and total n�mber
of cells in the presence of donors’ MSCs increased from
� x ��4 to �4 ± 4.� x��4� to 94 ± 7.7 x ��4 and to �95 ±
�4.7 x ��4 on day 7� �4 and �� respectively� p < �.��
and with patients’ MSCs from � x ��4 to �4 ± �.� x ��4�
to �5 ± �.� x ��4 and to �6� ± 7.� x ��4 on day 7� �4 and
��� respectively� p < �.��. No statistical difference was
fo�nd between effects of patients’ and donors’ MSCs
of on hematopoietic stem cells expansion.
To compare the f�nctional ability of donors’ and
patients’ MSCs to s�pport proliferation of CD�4+ cells�
we calc�lated the fold increase of cells n�mber �cells
n�mber at the o�tp�t/cells n�mber initial� for each
c�ltivation variant �Fig. ��. The greatest fold increase
was observed when CD�4+ cells were c�lt�red in me-
di�m containing GF complex on 7th day� with f�rther
increase on �4th and then ��st day. Active proliferation
was typical for CD�4+ cells in any c�ltivation variant.
The cells n�mber of CD�4+ cells from healthy donors
with GF alone increased from ��.�-fold to 74.6-fold
and to �55.� fold on 7th��4th and ��st day� respec-
tively� and for patients from ��.�-fold to �6.�-fold and
to ��6.4-fold on day 7th��4th and ��st� respectively.
While c�lt�ring cells with both GF and healthy donors
MSCs layer� hematopoietic cells n�mber increased
from �6.�-fold to ���.�-fold and to ���.5-fold on day
7th� �4th and ��st� respectively� and both GF and pa-
tients’ MSCs layer — from ��.9-fold to �5.�-fold and
to �6�.�-fold on 7th� �4th and ��st day� respectively.
On 7th� �4th and ��st day� the n�mber of c�lt�red
cells in the presence of both GF and MSCs layer was
significantly greater when compared with GF alone�
p < �.�5� both for healthy donors’ and patients’ MSCs.
The increase of the n�mber of hematopoietic cells in
the presence of donors’ MSCs layer compared with ab-
0
0,5
1
1,5
2
0 day 7 day 14 day 21 day
Ce
lls
x
1
04
0
40
80
120
160
200
0 day 7 day 14 day 21 day
Ce
lls
x
1
04
0
1
2
3
4
5
0 day 7 day 14 day 21 day
Ce
lls
x
1
04
0
40
80
120
160
200
0 day 7 day 14 day 21 day
Ce
lls
x
1
04
Donors
Patients
a b
c d
Cells Cells + GF
Сells + MSCs Сells + GF + MSCs
Fig. 2. Effects of several cytokines� and donors’ and patients’ MSCs on ex vivo expansion of hematopoietic stem cells. BM CD�4+ cells were
c�lt�red in fo�r c�ltivation variants: (a) Hematopoietic cells in ser�m-containing c�lt�re medi�m; (b) CD�4+ cells with GF. CD�4+ cells on
feeder layer of donors’ and patients’ MSCs; (c) CD�4+ cells with growth factors on feeder layer of donors’ and patients’ MSCs; (d) The res�lts
represent the mean cells n�mber ± SD of 6 different experiments on days 7� �4 and ��. MSCs of patients; MSCs of healthy donors
Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ��5
sence of MSCs layer was �6 vs ��.�-fold� respectively�
�p < �.��� on 7th day� ���.� vs 74.6-fold �p < �.��� on
�4th day� and ���.5 vs �55.�-fold on ��st day. C�lt�ring of
CD�4+ cells with patients’ MSCs layer also demonstrated
the significantly greater increase of hematopoietic cells
n�mber on 7th� �4th and ��st day as compared with
absence of MSCs layer:��.9 vs ��.�-fold� respectively�
�p < �.�5� on 7th day� �5.5 vs �6.�-fold �p < �.�5� on �4th
day and �6�.� vs ��6.4-fold �p < �.��� on ��st day.
0
50
100
150
200
250
7 day 14 day 21 day 7day 14 day 21 day
Fo
ld
i
nc
re
as
e
of
c
el
ls
Medium
GF
GF + MSC
* *
*
* *
*
*
*
*
*
* *
**
** **
**
**
Fig. 3. MSCs layer significantly increased proliferation of CD�4+
cells c�lt�red in medi�m with GF in comparison with CD�4+
cells grown in c�lt�re medi�m or c�lt�red in the presence of GF
alone. After 7th� �4th and ��st days cells were harvested� and
the fold increase was calc�lated. For statistical analysis� Mann —
Whitney U test was �sed to compare the effect of donors MSCs
and MSCs of patients on fold increase of hematopoietic cells
�*p < �.�5� **p < �.���
Statistical analysis �Mann — Whitney U test� com-
paring the fold increase in the n�mber of cells for two
independent gro�ps of donors and patients didn’t
detect statistically valid difference between the effects
on hematopoietic cells proliferation of co-c�lt�red for
� weeks donors’ MSCs and patients’ MSCs derived from
BM of patients with oncohematological diseases.
Influence of MSCs on changing of the cell
population profile after CD34+ cells expansion.
MSCs infl�ence on changing of CD�4+ cells pop�lation
profile after cells expansion was st�died by c�lt�ring
cells in liq�id LTC medi�m in presence of GF alone or
combined with MSCs for � weeks� �sing clonogenic
assay progenitors in methylcell�lose.
Morphological analysis of colony forming cells �CFC�
demonstrated the existence of pl�ripotent hematopoie-
tic prec�rsor cells CFU-GEMM� bypotential prec�r-
sors — CFU-GM and committing prec�rsors CFU-G�
CFU-M� BFU-E. All types of colony-forming �nits �CFU�
were scored and res�lts presented in Table. A mean
total of 579 CFC/��4 CD�4+ cells was observed in initial
samp les: ��% of cells ���5/��4 CD�4+ cells� corre-
sponded to CFU-G� �5% ��9/��4 CD�4+ cells� to CFU-
M� 4% ���/��4 CD�4+ cells� to CFU-GM� 49% ��6�/��4
CD�4+ cells� to BFU-E� and �.�% ��.6/��4 CD�4+ cells�
to m�ltipotent progenitor cells �CFU-GEMM�. A mean
total of �5� CFU/��4 CD�4+ cells was observed after ex-
pansion CD�4+ cells in c�lt�ral medi�m in the pre sence
of GF alone �pon a �-week c�lt�ring period. CFU com-
prised ��% of CFU-G� 6�% of CFU-M� 5.�% of CFU-GM�
�.�% of CFU-GEMM. When CD�4+ cells were grown in
the presence of the GF together with MSCs layer� the
spectr�m of CFU was: 4�% of CFU-G� 4�% of CFU-M�
��% of CFU-GM� �.6% of CFU-GEMM.
Table. Effect of MSCs on population profile of hematopoietic progenitors
throughout a 3-week culturing period
CD34+ cells
Progenitor cells per 1 x 104 CD34+ cells plated
(mean ± SEM)
CFU-G CFU-M CFU-GM CFU-
GEMM BFU-E
Initial cells (n = 9) 185 ± 73 89 ± 39 23 ± 17 0.6 ± 0.3 282 ± 119
Expansion with GF
(n = 7)
80.5 ± 35 155.5 ± 53 13 ± 6 0.7 ± 0.36 ND
Expansion with
GF+MSCs (n = 7)
113 ± 32 135.5 ± 64 31 ± 12 1.6 ± 0.7 ND
ND — not detected.
Th�s� o�r findings indicated that the dominant pop�-
lations of hematopoietic prec�rsors proliferated in vitro, in
initial samples and c�lt�red for �� days� were committing
prec�rsors CFU-G and CFU-M. The proportion of CFU-G/
CFU did not differ before and after CD�4+cells expansion
for �� days witho�t MSCs layer ���% vs ��%� or with
MSCs layer ���% and 4�%�. The proportion of CFU-M/
CFU increased after CD�4+cells expansion for �� days
witho�t MSCs layer from �5% to 6�% and with MSCs
layer from �5% to 4�% compared to CD�4+cells initial.
The proportion of immat�re CFU-GM and pl�ripo-
tent CFU-GEMM prec�rsors to total CFU increased af-
ter hematopoietic stem cells c�ltivation witho�t MSCs
layer and in the presence of MSCs compared to initial
samples: for CFU-GM from 4% to 5% and from 4% to
��%� respectively� and for CFU-GEMM from �.�% to
�.�% and from �.�% to �.6%� respectively.
Relative changes of progenitors n�mber in CD�4+
cell pop�lation after expansion for � weeks in presence
MSCs layer to n�mber of corresponding compartment
of CFU in initial samples of CD�4+ cells is shown on Fig. 4.
Relative changes of CFU-GM and CFU-GEMM n�mber
after c�ltivation both with MSCs and witho�t MSCs did
not differ significantly from n�mber of CFU-GM and
CFU-GEMM in initial pop�lation of CD�4+ cells. Tho�gh
n�mber of CFU-GM and CFU-GEMM were greater ap-
proximately by �.5-fold after expansion in the presence
of MSCs layer than witho�t MSCs �p < �.�5�. BFU-E
wasn’t detected in any analyzed sample after expansion
of CD�4+ cells �nder the described c�lt�ring conditions
for three weeks� while BFU-E n�mber consisted 49%
����.6 ± �59.�� in initial pop�lation of CD�4+ cells.
0
0.5
1
1.5
2
2.5
CFU-G CFU-M CFU-GM CFU-
GEMM
BFU-E
Fo
ld
in
cr
ea
se
c
om
pa
re
in
iti
al
c
el
ls Medium
GF
GF + MSC
*
*
Fig. 4. Relative changes of hematopoietic progenitors n�mber
after expansion of CD�4+ cells for �� days in presence of MSCs
layer. Corresponding compartments of CFU in initial samples of
CD�4+ cells recognized as � �n = 9; *p < �.�5�
��6 Experimental Oncology ��� �������� ���� ���ne�
DISCUSSION
In this work we examined the potential of MSCs
derived from the bone marrow of children with onco-
hematological diseases� treated by high-dose poly-
chemotherapy and radiotherapy� to s�pport hematopoi-
etic progenitors proliferation in vitro. It is important
for creation of a therape�tical strategy of application
of a�tologo�s MSCs for ne�tropenia period red�ction
after HSCs a�tologo�s transplantation for children with
ins�fficient n�mber of CD�4+ cells in graft.
In some p�blications it was demonstrated that BM
stroma greatly damaged after high-dose chemotherapy
or radiotherapy [��� ��]. Patients stromal cells c�lt�red
for 4�5 weeks after conditioning regimen� incl� ding
b�s�lphan and cyclophosphamide� are able to give
a monolayer only in ��% of cases in comparison with
��% shown for healthy donors [�4]. However� we �sed
proced�re of MSCs isolation and expansion from BM
of children with malignant neoplasms� and increased
initial n�mber of cells for more than � х ���-fold� as it
was demonstrated in o�r previo�s work [�5].
The ability of MSCs to s�pport hematopoiesis
in vitro has been shown by many a�thors in experi-
ments �nder the conditions of sim�ltaneo�s c�ltivation
of MSCs and HSCs [�6���].
Dexter et al were among the first researchers� who
st�died the infl�ence of stromal layer on proliferation
of BM cells. They had shown that stromal cells s�p-
ported hematopoietic cells proliferation and creation
of colony-forming �nit spleen �CFU-S�� while CFU-S
were absent when hematopoietic cells were c�ltivated
witho�t stromal layer [�9].
Some st�dies examined the capacity of c�ltivated
MSCs to s�pport h�man hematopoiesis in vitro. They
revealed that not only committed cells b�t also primi-
tive hematopoietic prec�rsors are able to proliferate
�nder the contact with MSCs. Th�s� hematopoietic
prec�rsors maintained the ability not only to differen-
tiate b�t also to self-replicate [��]. Wagner et al veri-
fied that primitive fractions of HPC had m�ch stronger
adherence to BM-derived MSCs than their more diffe-
rentiated co�nterparts� and that LTC-IC freq�ency was
higher in the adherent fraction than in the nonadherent
fraction of CD�4+ cells [��].
Koller et al and Breems et al demonstrated that
stromal cells significantly increased the n�mber of pro-
genitor and primitive stem cells after expansion of BM-
derived СD�4+ cells for � weeks in presence of IL-�� SCF�
GM-CSF� and erythropoietin. C�ltivation ex vivo red�ced
the ability of CD�4+ cells to prod�ce progenitors in LTC
witho�t stromal sol�ble factors or in presence of stromal
cells. The best expansion of CFU and LTC-IC and optimal
maintenance of graft q�ality were observed when PBSC
were c�lt�red in stroma-contact [�� ����4]. On the other
hand� Verfaillie et al demonstrated significantly worse
recovery of LTC-IC from c�lt�res� in which progenitors
were grown in contact with stroma. Also� researchers
have shown that direct contact with stroma inhibited
proliferation of LTC-IC even in case of gl�taraldehyde-
fixed stroma� which was no longer capable for prod�cing
growth inhibitory or stim�latory cytokines [�5]. At the
same time� Harvey et al showed that direct cell-to-cell
contact between HSCs and �rogenital ridge derived
stromal cells increased the CFU-C n�mbers 5 times
compared with non-contact co-c�lt�re [�6].
O�r observations of hematopoietic stem cells ex-
pansion in semisolid medi�m witho�t growth cytokines
in vitro testified that the presence of MSCs derived
from BM of patients with oncohematological disease
increased the n�mber of committing myeloid progeni-
tors approximately �-fold �p < �.�5�. The most intensive
proliferative activity of progenitor cells was shown in the
presence of patients’ MSCs and GF �SCF� GM-CSF� IL-��
and EPO�. In this case� we detected 7-fold increase in
the n�mber of myeloid progenitors and �4-fold in the
amo�nt of earlier pl�ripotent hematopoietic prec�rsor
cells �CFU-GEMM� compared with hematopoietic stem
cells expansion in methylcell�lose medi�m witho�t
MSCs and cytokines. These findings are in line with
data obtained for hematopoiesis s�pporting f�nction
of MSCs derived from BM of healthy donors� and with
statistical validity reveal that s�pporting effect on pro-
liferation of pl�ripotent hematopoietic prec�rsor cells
�CFU-GEMM� of patients’ MSCs did not differ from
s�pporting effect of MSCs from healthy donors.
In the present work we also st�died the effect of MSCs
layer from patients’ BM on maintaining of hematopoietic
stem cells proliferation in liq�id LTC medi�m containing
a combination of several cytokines. We revealed that the
presence of MSCs layer significantly promoted the rate
of hematopoietic cells proliferation on 7th� �4th and ��st
days� and total cells n�mber was m�ltiplied �6�.�-fold
on ��th day as compared with ��6.4-fold in the absence
of MSCs layer. Similar data was obtained when CD�4+
cells were grown on donors’ MSCs layer� and the cells
n�mber increased ���.5-fold in the presence of MSCs
layer compared to �55.�-fold witho�t MSCs.
Interestingly� o�r st�dy of hematopoietic stem cells
proliferation kinetics �nder vario�s c�lt�ring conditions
demonstrated that the presence of both patients’ and
donors’ MSCs layers witho�t GF didn’t have significant
infl�ence on expansion of hematopoietic cells within
a � week. At the same time� we showed that MSCs
from BM of both oncohematological patients and
healthy donors didn’t inhibit proliferation of commit-
ted progeni tors and primitive stem cells even if СD�4+
cells were c�ltivated in the absence of growth factors�
whereas in case of growing СD�4+ cells witho�t cytoki-
nes and MSCs we observed a loss of hematopoietic
cells. This fact confirmed the role of MSCs in preven-
tion of HSCs apoptosis [�7� ��].
It is very important that when CD�4+cells were c�l-
t�red both in semisolid medi�m or in liq�id LTC medi�m
the capacity of MSCs derived from BM patients with
oncohematological diseases to s�pport proliferation
and self renewal of hematopoietic prec�rsor cells
was the same as of MSCs derived from bone marrow
of healthy donors.
In the present work we st�died the cell pop�lation
profile �pon � weeks of CD�4+ cells expansion in LTC
Experimental Oncology ��� �������� ���� ���ne���� �������� ���� ���ne� ���ne� ��7
medi�m. Cells were c�ltivated with cytokines alone
or both cytokines and MSCs. We observed that MSCs
presence did not alter the pop�lation of committed
CFU-G and CFU-M prec�rsors as compared with initial
pop�lation of CD�4+ cells. The significant increase
in proportion of more primitive bipotent prec�rsors
�CFU-GM� and pl�ripotent �CFU-GEMM� prec�rsors
in the pop�lation of hematopoietic cells after CD�4+
cells expansion in the presence of MSCs layer is a very
important finding. Altogether� obtained res�lts are
in line with concl�sions made by other researchers
that stromal cells s�pport self-renewal of progenitor
compartment b�t they do not alter the differentiation
and proliferation of mat�re cells [��].
It is important that in o�r st�dy of prec�rsor cells
pop�lation grown for � weeks from isolated CD�4+
cells we observed a complete disappearance of
erythroid progenitors independently of MSCs layer
presence� whereas other a�thors� who st�died kinetics
of different hematopoietic progenitor cells in standard
long-term dextertype c�lt�res revealed a loss of sig-
nificant amo�nt of erythroid progenitors in 5 weeks
and the complete absence of these cells in 7 weeks.
The probable reason of disappearance of erythroid
progenitors in o�r st�dy co�ld be in the high amo�nt of
cytokines� s�ch as Flt-�-ligand� in the c�lt�ral medi�m�
which provided the shift towards myelopoiesis.
In s�mmary� we explored f�nctional potential of
МSCs derived from BM of children with malignancies
after high-dose chemotherapy or radiation to maintain
hematopoiesis in vitro and came to the concl�sion that
efficacy of patients’ MSCs to s�pport the proliferation
and self-renewal of hematopoietic cells was not diffe-
rent from MSCs derived from bone marrow of healthy
donors. In o�r st�dy all experiments with МSCs derived
from BM of patients with oncological disorders and
BM of donors were performed in identical conditions�
and both types of MSCs were derived and expanded
�sing common protocol. Moreover� a�tologo�s
MSCs of patients who were the candidates for HSCs
transplantation� similarly to donors’MSCs� greatly
increased the n�mber of more primitive bipotent mye-
loid and pl�ripotent prec�rsors� while co-c�ltivated
with hematopoietic stem cells. Th�s� we consider that
co-transplantation of a�tologo�s MSCs is good way
to improve hematopoietic stem cells engraftment and
red�ce a period of gran�locytopenia after a�tologo�s
HSCs transplantation in case of ins�fficient CD�4+ cell
n�mber in a�tologo�s transplant.
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ВЛИЯНИЕ МЕЗЕНХИМАЛЬНЫХ СТВОЛОВЫХ КЛЕТОК
ИЗ КОСТНОГО МОЗГА ДЕТЕЙ С ОНКОЛОГИЧЕСКИМИ
И ГЕМАТОЛОГИЧЕСКИМИ ЗАБОЛЕВАНИЯМИ
НА ПРОЛИФЕРАЦИЮ И СПОСОБНОСТЬ
К САМОПОДДЕРЖАНИЮ КЛЕТОК —ПРЕДШЕСТВЕННИКОВ
ГЕМОПОЭЗА IN VITRO
Цель: исследовали способность культивируемых in vitro мезенхимальных стволовых клеток (МСК), выделенных из костного
мозга детей со злокачественными новообразованиями, получавших в качестве лечения высокодозовую полихимиотерапию,
поддерживать гемопоэз in vitro. Материалы и методы: МСК выделяли и наращивали из проб костного мозга 8 пациентов
и 9 здоровых доноров детского возраста. CD34+ клетки выделяли из проб костного мозга доноров. Были поставлены
эксперименты по совместному культивированию МСК и CD34+ клеток в среде метилцеллюлозы и в среде для долгосрочного
культивирования с добавлением цитокинов и при их отсутствии. Результаты: в метилцеллюлозной среде наличие МСК
пациентов увеличивало количество коммитированных миелоидных предшественников в 2 раза, МСК совместно с ростовыми
факторами стимулировало пролиферацию этого типа клеток в 7 раз, а полипотентных предшественников — в 14 раз. При
долгосрочном культивировании с цитокинами на слое МСК общее количество ГСК на 21й день возростало в 161,2 раза. При
пролиферации CD34+ клеток в среде с МСК содержание бипотентных КОЕГМ повысилось с 4 до 11%, а полипотентных
КОЕГЭММ — с 0,1 до 0,6%. В экспериментах по экспансии CD34+ клеток результаты при использовании в качестве
фидерного слоя МСК пациентов или МСК доноров статистически не отличались. Выводы: проведенные исследования
позволяют сделать вывод о том, что МСК из костного мозга пациентов со злокачественными новообразованиями обладают
достаточным функциональным потенциалом для пролиферации и самоподдержания ГСК. Котрансплантация аутологичных
МСК при аутотрансплантации ГСК с низким содержанием CD34+ клеток в трансплантате может являться перспективной
для сокращения периода восстановления гемопоэза в ранний посттрансплантационный период.
Ключевые слова: мезенхимальные стволовые клетки, злокачественные новообразования, гемопоэз, предшественники
гемопоэза.
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