*5.2.1 MSC isolation procedure from bone marrow aspirates*

An effective BM-MSC injection is reliant on the performance of the marrow aspiration procedure, minimizing cellular trauma, while maximizing cellular yields and simultaneously avoiding peripheral RBC infiltration [62]. BM aspiration procedures, and not diagnostics, are routinely performed to collect bone marrow tissue to be processed using dedicated BM-MSC concentration kits for regenerative medicine applications. Kits may include a harvesting needle system and/or BM concentration device (**Figure 7**). These at POC MSC isolation techniques are a streamlined method to concentrate marrow cells, including MSCs, HSCs, and progenitor cells. These MSC centrifugation procedures demand less time and attention than laboratory preparation and culturing methodologies which are technically demanding. Double-spin centrifugation protocols create a layered BMC buffy coat stratum, based on different centrifugal forces that accomplish density cellular separation, as a result of the specific cellular gravity of the individual marrow components, as shown in **Figure 8**. Furthermore, BMA concentration-based technologies provide an economic and clinical/patient advantage when compared to the culturing technologies.

#### **Figure 7.**

*Regenerative Medicine*

*5.1.2 HSC and angiogenesis*

*lymphocytic cell variances.*

**Figure 6.**

regeneration [15, 59].

**5.2 Mesenchymal stem cells**

Emerging evidence suggests that BM-derived endothelial cells and HSCs, including their progenitor cells, contribute to tissue vascularization. HSCs deliver specific angiogenetic factors, facilitating the incorporation of endothelial progenitor cells into newly sprouting vessels. Several clinical studies have shown that BM-derived cells contribute to neo-angiogenesis during wound healing [44], critical limb ischemia [45], and postmyocardial infarction [58]. This should contribute to the clinical discussion of the value of BM-derived HSC and vascular progenitor as they are able to contribute to tissue restoration by accelerating tissue vascularization and

*Hematopoietic stem cell hierarchy. Self-renewing HSCs give rise to common myeloid progenitors and common lymphoid progenitors, producing different types of progenitor cells and ultimately fully differentiated cells. The myeloid progenitors produce granulocyte-macrophage progenitors giving rise to differentiated leukocytic cells and mast cells. The megakaryocyte/erythrocyte progenitors give rise to megakaryocytes, platelets, and erythrocytes. The lymphoid progenitors differentiate ultimately in* 

In recent decades, physicians performing regenerative medicine applications have been more interested in the potential of BM-MSCs than of HSCs. Imaginable reasons for this particular interest in MSCs might be recent published expert opinions: the *in vivo* ability of MSCs to migrate into tissues, their sturdy regenerative and reparative properties, and the MSC-mediated immunomodulatory actions.

**14**

*Bone marrow preparation essential components. In bone marrow concentration and preparation kits, the foremost components are a bone marrow harvesting needle and a concentration device (courtesy of EmCyte Corporation, Fort Myers, FL, USA).*

#### **Figure 8.**

*Bone marrow gravity separation following centrifugation. (A) Bone marrow aspirate in concentration device before centrifugation. In (B), the bone marrow aspirate is concentrated, with a view on the buffy coat stratum (gray layer on top of the RBC layer), referenced by the two black lines. Following a two-step centrifugation protocol, the centrifugal forces achieve density marrow cell separation due to the specific gravities of the individual marrow components.*
