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Bone Marrow Mesenchymal Stem Cells

Applications, isolation and culturing methods

Mesenchymal stem cells (MSCs) are stem cells that contribute to the regeneration of mesenchymal tissues1. They are multipotent cells that can replicate as undifferentiated cells and when induced have the potential to differentiate into cells of the mesenchymal lineage such as the bone, cartilage, muscle, ligament, tendon, adipose and stroma (Figure1)2.

The differentiation potential of MSCs can also extend into differentiating into cells of ectodermal and endodermal origin, such as hepatocytes3, neurons4, and cardiomyocytes5.

Multi-lineage potential of Bone Marrow derived MSCs

Figure 1 | Multi-lineage potential of Bone Marrow derived MSCs

Since the first description of MSCs by Friedenstein et al, in 1976 as clonal, plastic-adherent cells6, extensive research around MSCs has revealed the exceptional characteristics of this interesting type of adult stem cells.

MSCs can be extracted from varying sources, here we will stick to discussing MSC derived from the bone marrow. See this article we wrote for information on other sources.

MSCs within the bone marrow microenvironment are critical for supporting the growth and differentiation of e.g. primitive blood cells. They secrete various factors, that promote tissue repair, stimulate proliferation and differentiation7 of endogenous tissue progenitors, and modulate inflammatory and immune reactions.

This blog outlines the various therapeutic applications of bone marrow-derived MSCs (BM-MSCs), their isolation and culturing methods. Also described are quality control measures, including visual inspection criteria, that are useful for ensuring stemness i.e. self-renewal and multi-lineage differentiation potential.

BM-MSCs clinical applications

BM-MSCs are an attractive therapeutic tool due to their potential to be differentiated into a variety of cell types and their immunosuppressive properties. Their application is being explored in cell transplantation, regenerative therapy, correcting genetic disorders, and tissue engineering. Recent efforts focus on the use of BM-MSCs for the treatment of different diseases8, such as hematological disorders, diabetes, and autoimmune disorders9. Specific examples of disorders, belonging to various disease groups, where BM-MSC therapeutic potential is being evaluated, are given below (table 1).

Table 1 | Examples of various disease groups for which BM-MSCs therapies are being developed.

Bone and cartilage diseases

Cardiovascular diseases

Autoimmune disorders


Ischaemic heart failure

Crohn’s disease


Myocardial infarction

Graft versus host disease

Bone metastases

Renovascular hypertension

Multiple sclerosis

Osteogenesis imperfecta

Systemic lupus erythematosus

Knee joint cartilage defects

Rheumatoid arthritis

The positive outcomes from these MSC-based therapies are attributed to stimulation of several endogenous repair processes in injured tissues in vivo by secreted factors as well as modulation of the immune response. Cellular therapies using MSCs are based on either unselected BM-MSCs or specific sub-populations, which requires careful isolation of MSCs.

Bone marrow MSC isolation methods

Although MSCs have been isolated and studied from a number of different tissues, their most studied source has been the bone marrow (BM) primarily because of the wide differentiation potential of BM-MSCs. Nevertheless, since obtaining BM-MSCs can result in pain, bleeding or infection, there is an increasing interest in harvesting MSCs from other tissues such as peripheral blood10 or birth-derived tissues (umbilical cord).

Another concern associated with the use of BM-MSCs is that cell yield, longevity and potential for differentiation diminishes with donor age11. Compared with MSCs derived from other sources, BM-MSCs possess a longer duplication period, reach senescence earlier12 and constitute only 0.001–0.01% of nucleated marrow cells. Nevertheless, one advantage of BM-MSCs over other cell types is their relatively short culture time13.

Table 2 | Comparison of MSC sources - BM vs. umbilical cord


Umbilical cord MSC

High risk of contamination

Low risk of contamination

Invasive isolation procedure


Ready availability of source

Wait for umbilical cord to be delivered

Culture for 5 passages

Culture for 8-12 passages

Low doubling number

High doubling number

Requires relatively perfect HLA match

Greater HLA compatibility

One of the most interesting and practically useful properties of BM-MSCs, which is harnessed in the isolation and purification process, is their physical adherence to the plastic cell culture plate. Even though only a small percentage of cells in the bone marrow are MSCs, they are easily isolated14 from the other bone marrow nuclear cells because of this affinity to plastic.

In the absence of standardized isolation and culture expansion protocols for BM-MSCs, the way in which these cells are isolated, enriched, and cultured in vitro varies considerably between research groups. These varied tissue sources and methodologies of cell preparation beg the question of whether the resulting cells are sufficiently similar to allow for a direct comparison of reported biologic properties15 and experimental outcomes, especially in the context of cell therapy.

Most common methods used for isolation of MSCs are listed below.

BM-MSC isolation procedures typically use density centrifugation1 (with FicollTM, LymphoprepTM or PercollTM density mediums) to separate the mononuclear cell (MNC) fraction from the other marrow constituents (i.e. red blood cells, plasma, lipids) (Figure 2). This MNC fraction contains an enriched population of T-cells, B-cells, monocytes, hemopoetic stem cells (HSCs), endothelial progenitor cells and MSCs.

Following plating onto tissue culture flasks, MSCs, which represent the adherent cell population, form colonies. These cells grow most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm2)16 to generate single-cell derived colonies. For subsequent passages from a single cell colony, seeding densities that work best at enhancing proliferation and maintaining tri-lineage differentiation potential are 2000- 5000 cells/cm2 17. The cultures usually display a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase.

It is generally assumed that MSCs adhere within a few days after initial seeding and that the cell culture is rid of contaminating, non-adherent haemopoietic cells following serial media changes.

A variety of other techniques have been used for isolation and enrichment of MSCs, including antibody-based cell sorting18, low and high-density culture techniques19, positive negative selection method, frequent medium changes, and enzymatic digestion approach.

Figure 2 | Density centrifugation-based isolation of BM-MSCs (Adapted from Williams AR et al, DOI: 10.1161/CIRCRESAHA.111.243147)

To address the question of equivalence, regardless of the isolation method used, MSCs should fulfil certain minimal criteria15 laid down by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy. These universal criteria are important to ascertain the equivalence and stemness of MSCs isolated from different sources/donors and expanded from various protocols.

Summary of criteria to identify MSC

First, MSCs are plastic-adherent cells when they are maintained in standard culture conditions.

Second, they should express certain specific surface antigens. Surface antigens/markers are special proteins and carbohydrates present on the cell membrane that define cell type. BM-MSCs should express the surface markers CD105, CD73, CD90.

They should lack expression of CD45, CD34, CD14, CD11b, CD79a or CD19 and HLA-DR surface molecules as these are markers for other cell populations such as HSCs which are also a fairly abundant cell populations in the BM.

These surface antigens also facilitate the rapid identification and isolation of this cell population using flow cytometry and other related techniques.

Thirdly, when induced, BM-MSCs should be able to differentiate to osteoblasts, chondrocytes, and adipocytes in vitro.

Bone Marrow MSC Culturing methods

Even a small number of MSCs can multiply into millions of cells under the right culture conditions. Extensive protocols have been delineated for culturing stem cells in a variety of animals including humans.

The standard protocol for culturing and expansion uses medium consisting of Dulbecco’s Modified Eagles Medium (low glucose), supplemented with 10% fetal bovine serum (FBS) and L-glutamine at 37ºC with 5% CO2 at densities from 1 X 104 to 0.4 X 106 cells/cm2. Adherent monolayers are formed following culture for 6-7 days.

The cultures contain cells that self-renew rapidly. These rapidly renewing cells make up a large fraction of the cell population if the cultures are maintained at low density. In later passages, more mature cells, which have the lost the tri-lineage differentiation ability, start to predominate the cell population. It is therefore advisable to not expand BM-MSCs beyond five to ten passages.

Cultured MSCs are typically checked both morphologically and with respect to surface markers. MSCs in their undifferentiated state can be analyzed using bright-field or contrast microscopy. They appear as an adherent confluent monolayer of spindle-shaped cells that have a fibroblastic appearance (Figure 3). Cells become larger and less fibroblast-like when they start approaching senescence.

Figure 3 | Human BM-MSCs in culture (10X magnification), scale bar represents 100 mm. (Reprinted from Tucker D et al, DOI:

Visual evaluation of cultures of human MSCs by phase‐contrast microscopy (10X or 20X magnification) can be used to demonstrate changes in the morphology of the cells with time and with passage of the cells. The morphology of these cells changes as single‐cell‐derived colonies are expanded. In particular, small and rapidly self‐renewing cells that have the highest multipotentiality are gradually replaced by slowly replicating large, flat, and apparently mature, cells (mMSCs) - cells that have lost most of their multipotentiality. This transition to mMSCs20 is faster if cells are plated at high initial cell densities.

Confluence is another important characteristic to be considered as confluence at the time of harvest affects the properties21 of BM-MSCs which especially can affect their therapeutic potential. Growing MSCs to over confluence should be avoided since it is widely believed to lead to decreased cellular viability and differentiation potential. Human BM-MSCs typically reach confluency between 1 x 104 and 3 x 104 cells/cm2.


To follow BM-MSCs during the complete culturing period, follow the rate of proliferation and inspect any morphological changes, we recommend looking at the CytoSMART Lux2.


MSCs are known to express a large number of adhesion molecules (CD44, CD29, CD90), stromal cell markers (SH-2, SH-3, SH-4) and cytokine receptors [interleukin (IL)- 1R, tumor necrosis factor (TNF)-aR]. So along with removing contaminating HSCs by negative selection using antibodies to CD45, CD34 and CD11b, these MSC markers can be collectively used to help positively identify and isolate MSCs in culture.

Although visual evaluation and flow cytometric analysis can help investigators determine if the MSCs continue to display all their unique characteristics, the biologic property that most uniquely identifies MSC is their capacity for trilineage mesenchymal differentiation. So, investigators are encouraged to check for as many surface markers (positive and negative) as they deem important apart from demonstrating the trilineage differentiating potential of the MSCs.

Typically, around the third to fourth passage, the cultured cells can be harvested and transferred to specific induction media22 for facilitating differentiation into chondrocytes, adipocytes or osteocytes. Adipocyte and osteocyte populations usually form after 5-7 days in the differentiation medium (Figure 4), and chondrocytes appear after 14 days.

Figure 4 | Human BM-MSCs in culture: a. Spindle-shaped fibroblast-like appearance (20X), b. intracellular staining showing adipogenic differentiation, c. Osteogenic differentiation demonstrated by Alizarin red staining (5 days in differentiation medium, passage 2-3) (Reprinted from Baghaei K et al, Gastroenterol Hepatol Bed Bench 2017;10(3):208-213)

Concluding remarks:

No single surface marker is specific to the MSC population; thus, no single antibody can be used to isolate and enrich BM-MSCs from a mixed cell population. Therefore, combinations of various antibodies must be used in an attempt to characterize the MSC phenotype and allow their isolation.

Although BM-MSCs have been the main source of stem cells for many years, the inconvenience and risk of procuring these cells has driven research towards alternative and more practical sources.

Visual assessment by phase contrast microscopy can be used to evaluate the fibroblast-like morphology of the BM-MSCs, analyze their confluence, follow transitioning to mMSCs, and evaluate differentiation into the various lineages.

Learn about all CytoSMART imaging solutions here

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