Journal of ISSN: 2475-5540 JSRT

Stem Cell Research & Therapeutics
Short communication
Volume 1 Issue 6 - 2016
Characterization of Human Umbilical Cord Mesenchymal Stem Cells-Derived Conditioned Medium
Brian M Mehling1, Marine Manvelyan1*, Gabrielle Benesh1 and Dong Cheng Wu2,3
1Blue Horizon International, USA
2Wuhan University, China
3Department of Stem Cells, Wuhan Hongqiao Brain Hospital, China
Received: November 14, 2016| Published: November 17, 2016
*Corresponding author: Marine Manvelyan, Clinical Research Scientist, Blue Horizon International, LLC, 214 State Street, Hackensack, New Jersey 07601, USA, Tel: (201) 342-7662; Email:
Citation: Mehling BM, Manvelyan M, Benesh G, Cheng DW (2016) Characterization of Human Umbilical Cord Mesenchymal Stem Cells-Derived Conditioned Medium. J Stem Cell Res Ther 1(6): 00038. DOI: 10.15406/jsrt.2016.01.00038

Abstract

Medium where the stem cells are cultured is called conditioned medium (CM). In CM, Mesenchymal Stem Cells (MSCs) secrete different growth factors and cytokines with anti-inflammatory and anti-fibrotic effects. Numerous questions need to be answered before MSCs-derived CM can be used for the therapy of various diseases/conditions. The purpose of the present study is to analyze Human Umbilical Cord MSCs-derived CM for the presence of anti-inflammatory and regeneration-promoting cytokines. In our study of CM, immunoassay tests showed that all analyzed chemokines gave signals in the assays in positive control parallel wells. Thus all chemokines are functional and accurate to the indicated levels. Characterization of growth factors and cytokines in the MSCs-derived CM is crucial for further translation of CM in therapy of various diseases/conditions.

Keywords: Cytokines; Chemokines; Human umbilical cord mesenchymal stem cells; Conditioned medium

Introduction

Human umbilical cord mesenchymal stem cells (HUC-MSCs) are adult stem cells and a promising candidate for cell-based therapy [1]. HUC-MSCs therapy has advantages over human bone marrow MSCs because collection of HUC-MSCs is an easier, less expensive, non-invasive or painful method and there are no ethical considerations [2,3]. Medium where the stem cells are cultured is called conditioned medium (CM). In CM, MSCs secrete different growth factors and cytokines with anti-inflammatory and anti-fibrotic effects [4,5]. CM without the stem cells itself may cause tissue regeneration [6]. Stem cells derived CM, containing growth factors and cytokines, is a promising alternative that can overcome the poor engraftment of the transplanted stem cells and the potential risk of cancer development [7]. CM can be manufactured, freeze-dried, packaged, and transported more easily than stem cells [8]. Numerous questions need to be answered before MSCs-derived CM can be used for the therapy of various diseases/conditions. Analysis and characterization of cytokines and growth factors in CM is a very important step in the translation process of CM usage. The purpose of the present study is to analyze HUC-MSCs derived CM for the presence of anti-inflammatory and regeneration-promoting cytokines.

Materials and Methods

Preparation of UC-MSCs and isolation of MSCs from umbilical cord bloods

Umbilical cord bloods were collected and MSCs were isolated from umbilical cord blood according to methods described by Mehling et al. [9].

Preparation and culture of CM

The supernatant was discarded and the cell pellet was re-suspended in the basic medium (GIBCO AIM V® Serum Free Medium). The cells were cultured in GIBCO AIM V® Serum Free Medium for 14 days at 37ËšC. Fresh medium was added to the cultured cells every two days. After 14 days of cell culture, the cells were harvested by centrifuge and supernatant medium was collected. The medium was filtered by 0.22um membrane. Germ test showed that the maximum of germs is limited for cosmetic products. Medium was frozen at -80ËšC.

Cytokine immunoassay

Bio-Plex Pro Human Chemokine 40-plex Panel was used to detect and characterize 40 chemokines. Cytokine Immunoassay was performed according to the manufacturer instructions (Bio-Plex Pro™ Human Chemokine Panel).

In order to characterize chemokines in CM, the assays were done using two separate experiments, with each sample tested three times within each experiment. The control media was without conditioning

Results

Previous studies showed that CM have been tested in various kinds of diseases/conditions, including alopecia, acute and chronic hind limb ischemia, acute and chronic wound healing, spinal cord injury, lung injury and others. Application of CM, containing various cytokines, showed improvement of these conditions [8,10].

In our study of CM, immunoassay tests showed that all analyzed chemokines gave signals in the assays in positive control parallel wells. Thus all chemokines are functional and accurate to the indicated levels. Statistically significant difference was revealed between control group and CM (Table 1). Statistical analysis was done with the application of statistical software package Sigma Plot 12.0.

Cytokine

Unit

Control Average ± Standard Error

Cm Average ± Standard Error

Statistical Significance

CCL21

pg/mL

30 ± 12

485.5 ± 5.5

YES, P = <0.001

CXCL13

pg/mL

18 ± 0

171.5 ± 3.5

YES, P = <0.001

CCL27

pg/mL

15 ± 3

603.5 ± 8.5

YES, P = <0.001

CXCL5

pg/mL

208.5 ± 9.5

7458.5 ± 133.5

YES, P = <0.001

CCL11

pg/mL

11.5 ± 2.5

101 ± 4

YES, P = <0.003

CCL24

pg/mL

14.5 ± 9.5

332.5 ± 7.5

YES, P = <0.001

CCL26

pg/mL

8.5 ± 3.5

61.5 ± 3.5

YES, P = 0.009

CCL19

pg/mL

25 ± 3

73 ± 9

YES, P = 0.037

CX3CL1

pg/mL

36.5 ± 6.5

749 ± 17

YES, P = <0.001

CXCL6

pg/mL

36.5 ± 1.5

232 ± 9

YES, P = 0.002

GM-CSF

pg/mL

26.5 ± 2.5

48 ± 3

YES, P = 0.031

CXCL1

pg/mL

13 ± 1

85 ± 3

YES, P = 0.002

CXCL2

pg/mL

21.5 ± 7.5

503.5 ± 11.5

YES, P = <0.001

IFN-gamma

pg/mL

19.5 ± 2.5

50 ± 2

YES, P = 0.011

IL-1b

pg/mL

8.5 ± 3.5

78 ± 5

YES, P = 0.008

IL-2

pg/mL

2.5 ± 0.5

15 ± 1

YES, P = 0.008

IL-4

pg/mL

4.5 ± 0.5

222 ± 3

YES, P = <0.001

IL-6

pg/mL

25.5 ± 3.5

45 ± 3

NO*, P = 0.052

IL-8

pg/mL

10.5 ± 2.5

60.5 ± 2.5

YES, P = 0.005

IL-10

pg/mL

36 ± 16

180.5 ± 4.5

YES, P = 0.013

IL-16

pg/mL

26.5 ± 1.5

333.5 ± 8.5

YES, P = <0.001

CXCL10

pg/mL

11 ± 1

170 ± 1

YES, P = <0.001

CXCL11

pg/mL

15.5 ± 0.5

184 ± 4

YES, P = <0.001

CCL2

pg/mL

19 ± 0

211.5 ± 6.5

YES, P = <0.001

CCL8

pg/mL

1.5 ± 1.5

39 ± 3

YES, P = 0.008

CCL7

pg/mL

2.5 ± 0.5

15 ± 3

NO*, P = 0.054

CCL13

pg/mL

0.5 ± 0.5

14.5 ± 0.5

YES, P = <0.001

CCL22

pg/mL

44.5 ± 3.5

533 ± 9

YES, P = <0.001

MIF

pg/mL

13.5 ± 1.5

167 ± 6

YES, P = 0.002

CXCL9

pg/mL

11 ± 1

151 ± 3

YES, P = <0.001

CCL3

pg/mL

7 ± 1

33.5 ± 1.5

YES, P = <0.001

CCL15

pg/mL

1.5 ± 0.5

11 ± 1

YES, P = 0.014

CCL20

pg/mL

3 ± 0

12 ± 2

YES, P = 0.046

CCL19

pg/mL

18 ± 3

154 ± 4

YES, P = <0.001

CCL23

pg/mL

2 ± 2

37.5 ± 3.5

YES, P = 0.013

CXCL16

pg/mL

34 ± 4

1205 ± 25

YES, P = <0.001

CXC12

pg/mL

59 ± 3

1861 ± 14

YES, P = <0.001

CCL17

pg/mL

10.5 ± 1.5

124 ± 4

YES, P = <0.001

CCL25

pg/mL

80.5 ± 11.5

1911 ± 29

YES, P = <0.001

TNF-alpha

pg/mL

8.5 ± 3.5

52 ± 8

YES, P = 0.038

Table 1: Chemokine assay results.

*The calculated P is not much higher than P=0.05

Different factors in CM act together to promote regeneration. Proinflammatory cytokines (for example IL-1β, IL-6, and TNF-α) are involved in the up-regulation of inflammatory reactions.

Chemokines are functionally divided into two groups: 1) Homeostatic chemokines (for example CCL19, CCL20, CCL21, CCL25, CCL27, CXCL12, CXCL13) are responsible for basal leukocyte migration; 2) Inflammatory chemokines (for example: CCL2, CCL3, CCL11, CXCL10, CXCL1) are formed under pathological conditions and actively participate in the inflammatory response attracting immune cells to the site of inflammation.

Anti-inflammatory cytokines (for example: IL-1, IL-2, IL-10) are a series of immunoregulatory molecules that control the pro-inflammatory cytokine response. Stem cell derived CM therapy is a rapidly advancing field that promises to have a substantial impact on the treatment of different diseases/conditions. Therefore, gaining a more complete characterization of growth factors and cytokines in the MSCs-derived CM is crucial for further translation of CM in therapy of various diseases/conditions.

References

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